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Fiziologia-- Physiologysupplement
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Fiziologia- Physiology 2012.22.1(73)
Fiziologia- Physiology 2010supplement

CONTENTS

1. Interactions between Mesenchymal Stem Cells and Cellular Effectors of Immune System
Laura Marusciac, Carmen Panaitescu, Virgil Paunescu ................................................................................................................................................................................................. 4
2. Relationship between Serum Levels of Interleukin-2 and Liver Injury in Chronic Hepatitis C Virus Infection before Interferon Treatment
Manole Cojocaru, Simona Alexandra Iacob, Dorina Banica, Eugenia Panaitescu, Diana Gabriela Iacob ....................................................................................................................11
3. Therapeutic Particularities in Adult Patients with Thyroid Disease and Diabetes
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas . ............................................................................................................................................................................................13
4. Peloidotherapy Modulates Cortisol and Thyrotrophin-Stimulating Hormone Balance
Surdu Traian-Virgiliu, Ion Ileana, Surdu Monica . ........................................................................................................................................................................................................20
5. Myasthenia Gravis: Cardiac and Pharmacological Considerations
Bogdan Istrate, Crisanda Vilciu, Maarten Hulsmans, Carmen Tatu .............................................................................................................................................................................24
6. REVIEW: Liver and Immunity
Suciu Maria, Scurtu Ileana, Ardelean Aurel . ...............................................................................................................................................................................................................27
7. Prevalence of Carotid Artery Stenosis and Myocardial Ischemia in Patients with Peripheral Arterial Disease
Gruici A, Ardeleanu E, Mihăescu R, Matusz AA, Alaman R, Gurgus D, Gâlcă E ...........................................................................................................................................................32

CUPRINS
1. Interactinea dintre celulele stem mezenchimale si efectorii celulari ai sistemului imun
Laura Marusciac, Carmen Panaitescu, Virgil Paunescu ................................................................................................................................................................................................. 4
2. Relatia dintre nivelurile serice ale interleukinei-2 si distrugerea hepatics in infectia cronica cu virusul hepatitei B
Manole Cojocaru, Simona Alexandra Iacob, Dorina Banica, Eugenia Panaitescu, Diana Gabriela Iacob ....................................................................................................................11
3. Particularitati terapeutice ale pacientilor adulti cu afectiuni tiroidiene si diabet
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas . ............................................................................................................................................................................................13
4. Peloidoterapia moduleaza echilibrul cortizonic si al TSH
Surdu Traian-Virgiliu, Ion Ileana, Surdu Monica . ........................................................................................................................................................................................................20
5. Miastenia gravis: consideratii cardiace
Bogdan Istrate, Crisanda Vilciu, Maarten Hulsmans, Carmen Tatu .............................................................................................................................................................................24
6. REVIEW: Ficatul si imunitatea
Suciu Maria, Scurtu Ileana, Ardelean Aurel . ...............................................................................................................................................................................................................27
7. Prevalenta stenozei de artera carotida si a ischemiei miocardice la pacientii cu boala arteriala periferica
Gruici A, Ardeleanu E, Mihăescu R, Matusz AA, Alaman R, Gurgus D, Gâlcă E ...........................................................................................................................................................32

2012.22.1 (73)  Fiziologia - Physiology

3

INTERACTIONS BETWEEN MESENCHYMAL STEM CELLSAND CELLULAR EFFECTORS OF IMMUNE SYSTEMLAURA MARUSCIAC, CARMEN PANAITESCU, VIRGIL PAUNESCU
Department of Functional Sciences, „Victor Babes“ University of Medicine and Pharmacy Timisoara

ABSTRACT
Mesenchymal stem cells represent a rare subset of stem cells that reside in the bone marrow, where they interact with hematopoietic
stem cells, guiding their growth and differentiation, and they serve as a reservoir of renewal of various mesenchymal tissues. They
also possess remarkable immunosuppressive properties, inhibiting the proliferation and function of most major immune cell populations. This review provides information regarding the current research on the interactions of mesenchymal stem cells with immune
cells, the possible mechanisms of these interactions, the in vivo use outcomes, as well as other potential clinical implications, andfuture research directions.
Key words: mesenchymal stem cells, immune system, immunosuppression, clinical applications

INTRODUCTION
In vitro interactions of mesenchymal stem cells (MSCs) withmultiple sets of immune cells have shown that MSCs possess
remarkable immunosuppressive properties, being able to inhibit
the proliferation and modulate the function of the major immune
cell populations, including T cells, B cells and natural killer (NK)cells. They have also been shown to modulate the activitiesof dendritic cells (DCs) and to induce regulatory T cells. The
immunomodulatory effect of MSCs is mediated by non-specificanti-proliferative mechanisms, dependent on cell–cell contact orsecreted soluble factors.
These unique properties make MSCs ideal candidates
for clinical application as immunosuppressants, in pathologies
such as graft-versus-host disease, solid organ transplants, or
autoimmune diseases.
MSCs and T cells
The correlation between MSCs and T cells starts in theearly ontogenic stage, both in the bone marrow (BM) and in
the thymus. MSCs derived from the mesothelium invade thevasculature and localize in the BM stroma, where they provide
support for the hematopoietic stem cells [1]. Primitive T cells
migrate to the thymus, where they undergo positive and negative selection, leading to their maturation. It has been shown thatmarrow stromal cells can support T-cell maturation even in theabsence of the thymus, suggesting that the bone marrow stroma
can function as an extrathymic site for T cell development [2].

This leads to the conclusion that MSCs may play an importantrole in the process of hematopoiesis.
In vitro and in vivo studies have shown multiple effects of
MSCs on T cells. However, they differ, depending on the concentration of the MSCs. A high MSC/lymphocyte ratio is associatedwith an inhibitory effect of MSCs, while a low MSC/lymphocyte
ratio is often accompanied by enhanced T cell proliferation [3].
MSCs may have dual roles, depending on the environment towhich they are exposed. They have been shown to inhibit the
proliferation of activated T cells, but they can also support thesurvival of quiescent T cells (as they are in the hematopoietic
stem cell niche) [4]. Under quiescent conditions, MSCs canpromote T cell survival, and stimulate CD4+ T cell activation andproliferation, independently of cell-cell contact [5].
Some of the mechanisms of immunosuppression havebeen elucidated, but the underlying molecular mechanisms
remain uncertain. This immune inhibition seems to be mediatedby cell-cell interactions, but also by the release of cytokines [6].
Apparently, species-dependent mechanisms are involved: inrodents cell-cell contact is enough [3], but in humans soluble
factors are necessary for the suppressive effect [6].
MSCs can inhibit T cell proliferation by different stimuli, suchas alloantigens, cognate antigen stimuli, and non-specific mitogen stimuli. The inhibition affects different aspects of proliferation,such as the expression of activation markers, cytotoxic T (Tc) cell
formation (they can also inhibit the cytotoxic effects of antigen-

Received 20th of December 2011. Accepted 15th February 2012. Address for correspondence: Laura Marusciac, MD PhD student, Department of Functional Sciences,„Victor Babes“ University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No.2A, RO-300041, Timisoara, phone/fax: +40256220479; e-mail: laura.
marusciac@gmail. com

4

Fiziologia - Physiology  2012.22.1 (73)

primed Tc cells), cytokine production (interferon (IFN)-γ by Th1
cells, interleukin (IL-4) by Th2 cells) [7]. The suppressive effectis present on both naïve and memory T cells, CD4+ and CD8+
T cells [3]. It does not require major histocompatibility complex
restriction and can be mediated by allogeneic MSCs [8]. The
accumulation of cells in the G0/G1 phase of the cell cycle may
mean that MSCs act through the inhibition of cell division [9].
Several MSC-derived molecules have been proposed which
are believed to exert immunomodulatory effects on T cells: transforming growth factor (TGF)-β1 [10], hepatocyte growth factor(HGF) [3], indoleamine 2, 3-dioxygenase (IDO) [5], prostaglandin
E2 (PGE2), and human leukocyte antigen-G5 (HLA-G5) [3].
MSCs treated with IFN-γ express functional IDO, which
degrades tryptophan, resulting in kynurenine synthesis, which
in turn suppresses lymphocyte proliferation [11].
If MSCs are co-cultured with T cells, a high level of PGE2
can be found in the medium, and treatment with PGE2 inhibitors reduces the MSC-mediated immune modulation [12]. The
mechanisms of this process are still uncertain.
The secretion of human leukocyte antigen-G5 (HLA-G5) by
MSCs is believed to be essential for the suppression of T celland NK cell function, the shift of the allogeneic T cell response
towards a T helper type 2 (Th2) cytokine profile, and the induction of CD4+CD25highforkhead box P3 (FoxP3+) regulatory T
cells (Tregs) [3].
MSCs may also function through suppressive T cells.
Apart from Th cells and Tc cells, there is a third type of T cells– the regulatory T cells, which negatively regulate immune
responses. Three subsets of Tregs have been identified so far:CD41+CD25highfoxP3+ (naturally occurring, acting in an antigennon-specific manner), CD4+CD25+/-, and CD8+CD28- (both are
induced, and they both act in an antigen-specific manner) [13].
MSCs and B cells
B cells originate in the bone marrow and are in close contact
with marrow stromal cells during their development. After going
through negative selection and clonal deletion, the majority ofthem (in mice, 90%) die without ever reaching the circulating
pool [1]. The negative selection process takes place with thehelp of stromal cells, through a classic immune cell-mediatedmechanism. Self-antigens present on stromal cells cross-link
self-antibodies of mature B cells, leading to their negative selection. MSCs have been involved in the negative regulation of B
cell development through an additional mechanism: the LY-6A/E
protein (or Sca-1, present on MSCs) increases GM-CSF production, which inhibits B cell development [14].
The B cell inhibition seems to be mediated mostly by cell-cellinteractions, but also by the release of soluble cytokines, whichleads to the arrest of the cell cycle in the G0/G1 phase [6].
MSC-derived molecules can inhibit B cell proliferation anddifferentiation, precluding the necessity of HLA matching of donor and host [15]. They have also been shown to downregulate
chemokine receptors on B cells, decreasing their migration to
inflammation sites [16].
MSCs can inhibit the proliferation and differentiation into
2012.22.1 (73)  Fiziologia - Physiology

antibody-secreting cells of B cells activated with anti-Ig antibodies, soluble anti-CD40L antibody and cytokines (IL-2, IL-4), while
also impairing their chemokine receptor (CXCR4, CXCR5 andCCR7) production and secretion [3]. These are receptors for
CXCL12 and CXCL13, which are chemokines responsible forchemotaxis, and playing an essential role in B cell positioning
in secondary lymphoid organs. MSCs were unable to affect theexpression of other cytokines and co-stimulatory molecules by
the B cells [16].
This inhibition process took place only in the presence ofIFN-γ (produced by activated B cells) [17], leading to the conclusion that IFN-γ causes MSCs to produce IDO, which suppresses
lymphocyte proliferation through the tryptophan pathway, throughan uncertain mechanism.
As with T cells, MSC have different actions on B cells,
depending on their concentration: low numbers of MSCs havea stimulatory effect, while MSCs in excess are immunosuppressive. A possible explanation might be that the stimulatory
pathway becomes overloaded when the MSCs are in excessive
quantities [10].
MSCs can also assist the estrogen and androgen suppressive effects on B cells. The effect of androgen is mediated byandrogen receptors expressed on the MSC surface [1].
TGF-b produced by MSCs has also been shown to inhibit
B-cells, through the downregulation of IL-7 [18].
However, there is still no in vivo evidence of the suppressive
effect of MSC on B cells.
MSCs and NK cells
As with T and B cells, NK cell progenitors originate in theBM, where they establish close interactions with marrow stromalcells [19].
The molecular mechanisms underlying the immunosuppressive effects of MSCs on NK cells are still uncertain.
Different studies have shown variable results. Some haveshown that IL-2 and IL-15-induced NK cell proliferation and IFN-γ
production are inhibited by MSCs [20], but according to others
MSCs only partially inhibit the proliferation of activated NK cells[21], or they are lysed by IL-2-activated NK cells [22].
The MSC effect on NK cell toxicity seems to differ depending on experiment set-ups. For example, there was no inhibitionof NK cell-mediated lysis when freshly isolated NK cells wereused in order to lyse allogeneic, HLA class I negative or positive
targets, in the presence of MSCs [21]. However, when cultured
NK cells were used (4-5 days culture, with IL-2) to lyse K562
cells in the presence of MSCs, they were less efficient than NK
cells that were not exposed to MSCs [23]. This suppressive effectmight be due to the IFN-γ produced by NK cells.
Allogeneic and syngeneic MSCs are vulnerable to lysis byactivated NK cells, due to their low expression of HLA class I
molecules and to the MSC expression of several ligands, such as
ULBP, PVR and nectin-2, which are recognized by activated NK
cell receptors (NKp30, NKG2D and DNAM-1) [3]. If the MSCs are
incubated with IFN-γ, the HLA expression is up-regulated, leading
to a decreased MSC susceptibility to NK cell-mediated lysis [19].
5MSCs are also capable of inhibiting the surface expression of NK
cell receptors, such as NKp30 and NKG2D, which are involvedin NK cell activation and target cell lysis [6].
MSCs may exert an effect on NK cell cytokine production.
After co-culture with MSCs, IL-2-activated NK cells produced
decreased amounts of IL-15-induced cytokines – IFN-γ, IL-10,
TNF-α [19].
Like the case with T or B cells, immunosuppressive soluble
molecules, such as PGE2, IDO, or TGF-β1 (less important), havealso been proposed to play a role in the inhibition of NK cells [19].
Synthesis of IDO in MSCs seems to be induced both directly(through MSC exposure to IFN-γ), and indirectly, after autocrine
stimulation with PGE2, which induces de novo expression of IDO
in MSCs. Moreover, IFN-γ and TNF-α, secreted by NK cells afterIL-2 activation, may induce the production of PGE2 [24].
MSCs and DCs
Dendritic cells (DCs) have an essential role in naïve T cellstimulation during the primary immune response. They are also
involved in the activation of B cells, either directly through soluble
molecules, or indirectly through Th cells. This means that DCs are
critical to both cell-mediated immunity and humoral immunity.
MSC-induced inhibition of DCs seems to be mediated bysoluble molecules, such as PGE2 [25], released upon cell-cell
contact.
MSCs are able to inhibit the differentiation, maturation andactivation of co-cultured DCs. They can inhibit the differentiationof monocytes to DCs [26], downregulating the expression of several differentiation markers, such as CD1a, CD86 and HLA-DR[4], and they can also inhibit the maturation of DCs, through thedownregulation of CD83 expression, deflecting their phenotype
towards an immature status [1]. The immature DCs secrete lower
amounts of TNF-α, and higher amounts of IL-10 [12].
DCs may also be involved indirectly in the immunosuppression of T cells by MSCs, through several different possible
mechanisms. MSCs decrease the IL-12-secreting capacity ofDCs, which is essential in cell-mediated immunity, where it
activates naïve T cells and differentiates them towards a Th1
phenotype [27]. MSCs also secrete TGF-β1, which is a soluble
factor able to inhibit DC activation and maturation. Also, DC
maturation may be delayed through the preferential activationof CD4+CD25+ Treg cells [28]. MSCs may also lead to a stateof immunotolerance through the decrease of TNF-α secretionin mature DC1s, and through the increase of IL-10 secretion inmature DC2s. IL-10 has an inhibitory effect on the functions ofantigen presenting cells, downregulating the IL-12 secretion, andthe expression of several surface markers, such as CD40, CD80,
CD83 and CD86. Therefore, DCs that secrete IL-10 have little
or no stimulatory effect when co-cultured with T cells, inhibiting
their proliferation [1].
MSCs have been shown to impair the differentiation of
monocytes into DCs by inhibiting their response to maturation signals, and decreasing their expression of co-stimulatory molecules[27]. This inhibitory effect may be mediated by soluble molecules,
and it may be dose-dependent. After co-culture with MSCs, the6cell cycle of DCs was arrested in the G0/G1 phase [28].
MSCs have different effects on different DC populations.
On myeloid DCs, the effect is to induce a decreased productionof TNF-α, while on plasmacytoid DCs the effect is to increase
IL-10 production, leading to decrease IFN-γ by Th1 cells, increased IL-4 secretion by Th2 cells, and an increased numberof Tregs [19].
A scheme of the main important interactions between MSCs
and the cells of the immune system can be seen in Figure 1.

Fig.1. Immunomodulatory effects of MSCs [25]MSCs and GVHD
GVHD (graft-versus-host-disease) is a condition that causes
considerable morbidity and mortality after HSCT (hematopoietic
stem cell transplantation). The main targets for acute GVHD are
the skin, the gastrointestinal tract, and the hematopoietic system.
If the graft is T-cell depleted, the GVHD is reduced, but whenusing non-manipulated grafts, it needs to be prevented withimmunosuppressive drugs, such as cyclosporine, methotrexate, high-dose steroid therapy, anti-IL-2 receptor antibodies,monoclonal anti-CD3 antibodies, anti-TNF-α antibodies, andothers. There are different grades of GVHD, and grade IV islife-threatening [29].
During the past 15 years, there have been promising resultsin patients treated with MSCs. Patients with HSCT undergoing
MSC infusion have shown significantly improved clinical outcomes without significant adverse effects [30]. Also, all patients
who received MSCs have shown sustained hematopoietic engraftment, and no adverse reactions [26]. When there is HLA disparity in the donor/recipient pair, it seems that co-transplantationof HSCs (hematopoietic stem cells) and MSCs modulate the hostalloreactivity, and promote better cell engraftment.
Where GVHD prophylaxis is concerned, study results
have shown that the co-transplantation of HSCs and third-partyor same-donor MSCs is safe and that it reduces nonrelapse
mortality [31]. These results should be interpreted with caution,
though, due to the small numbers of subjects and the lack ofcontrol cohorts. Also, further studies are required to investigate
the MSC effects on GVL (graft-versus-leukemia).
Chronic GVHD is another major cause of morbidity andmortality after allogeneic HSC. It has been shown that MSCs
may ameliorate sclerodermatous chronic GVHD, as well as the
Fiziologia - Physiology  2012.22.1 (73)

cutaneous, hepatic, and gastrointestinal manifestations of thedisease [32]. Further research is needed in order to establish
whether the GVL effect is preserved in patients with MSC-treatedchronic GVHD.
There are still on-going clinical trials trying to establish
the number of infusions to be performed, the optimal dose of
MSCs per infusion, and the possible interactions with othertherapies.
Besides the concerns regarding the preservation of the
GVL effect, there is also concern that MSCs may function as a
sanctuary for leukemic blast cells, promoting disease relapse[31]. Apoptosis has been shown to be inhibited in leukemic cells
that interact with MSCs, which also transiently arrest the cell cycleof hematopoietic and non-hematopoietic tumor cell lines [33].
One should also keep in mind the possibility of tumorigenicity,
ectopic activity, infectious agent transfer, and other potential
adverse effects.
Further research is needed, both preclinical and correlativein the context of carefully controlled trials, to help address theseimportant issues.
MSCs and solid organ transplants
Organ transplantation has become standard therapy in suchcases as kidney, heart, or liver failure, being one of the mostremarkable achievements in modern surgery. Even though organtransplants are highly successful, there usually is ongoing needfor immunosuppression, which results in significant morbidity due
to infections and malignancies.
The goal of immunosuppression in solid organ transplants isdifferent than in HSCT, aiming to prevent the immune responseof the host organism against the graft. It is not necessary tobalance this against the graft-versus-tumor effect. MSCs may
be more effective at allowing specific targeting of the immunoinhibitory effect. A key difference between HSCT and solid organtransplant rejection is that in solid organ transplantation there
is also involvement of immune humoral responses. Hyperacutegraft rejection has become rare since the introduction of routine
crossmatching before transplantation, but antibody-mediatedrejection is still an important cause of acute and chronic allogeneic graft dysfunction.
Animal studies have shown that MSCs migrate to cardiac
allogeneic heart grafts in patients with chronic graft rejection [26],delivering localized immunosuppression, and thereby minimizing
non-specific, systemic immunosuppressive complications. For
example, a single dose of intravenous donor MSCs had a modest, but significant improvement in a baboon skin graft model[34], while systemic administration of MSCs in a rat cardiac
allograft model also had a significant positive effect on survival[35]. However, in another mismatch cardiac model, no MSC effecthas been found on allograft outcomes [36]. In the majority of thestudies allogeneic MSCs were co-infused at the time of organtransplantation. The reasons for these differences are uncertain,
but improved survival was not associated with tolerance in thestudies, suggesting that MSCs alone are probably not sufficiently
immunosuppressive for vascularized transplants.
2012.22.1 (73)  Fiziologia - Physiology

Another potential use for MSCs might be to promote a stateof immunological chimerism, as well as long-term graft tolerance
by the host immune system, after co-infusion of MSCs at the
moment of the solid organ transplantation. When immunologic
chimerism is established (through simultaneous transplantationof solid organ and bone marrow from the same donor), there islong-term graft survival in the absence of immunosuppression[37].
Most of the immunomodulatory functions and propertiesof MSCs have been shown in vitro, and further animal studies
are necessary in order to evaluate their potential use in clinicalsolid organ transplantation. However, despite the lack of clinicalstudies, MSCs remain attractive for the therapy of solid organ
rejection, because of their immunoregulatory effects on the host,promoting graft tolerance, their trophic functions, which help tominimize ischemic and inflammatory injuries to the graft.
MSCs and autoimmune disease
MSCs have also been considered for the therapy of autoimmune diseases. To that purpose, they have been tested in various
animal models of autoimmune diseases, such as experimental
autoimmune encephalomyelitis [38], diabetes [39], systemic
lupus erythematosus [40], and rheumatoid arthritis [41]. Autoimmune diseases involve aberrant recognition of host tissues by
the immune system, which mounts an immune response against
its own tissues. The clinical manifestations affect many organs,such as the central nervous system, pancreas, joints, or multiple
systems (like in systemic lupus erythematosus). Even though theunderlying immunopathological causes are similar, one cannot
always extrapolate from one disease to others.
Mice with experimental autoimmune encephalomyelitis(EAE) constitute a model for human autoimmune diseases.
Mice that received MSCs at the onset and at the peak of thedisease showed lower levels of demyelination, axonal loss, andinflammation than control mice, and their proliferation of T cells
from the spleen and lymph nodes was reduced, as well as the
production of proinflammatory factors (TNF-α and IFN-γ). MSCs
were able to improve disease symptoms, as well as to decrease
relapses. The therapeutic effect was maximal when the MSCs
were administered at the onset of the disease, but there wasno effect after disease stabilization [38]. These findings could
not be replicated in a clinical study on patients with multiple
sclerosis who had received a single dose of intrathecal MSCs.
Some patients showed some clinical neurological improvement,
but this could not be documented at radiological investigations.
Only one of the patients showed a quantitative improvementin the central nervous system plaque, on magnetic resonanceimaging [42].
In type II collagen-induced arthritis, administration of allogeneic MSCs prevented the irreversible immune destructionof cartilage and bone [43]. However, another study showed that
MSCs in fact worsened the clinical parameters [26]. A possible
reason for this is that MSCs were administered at different times
after disease induction. As MSCs can also be used in bone andcartilage replacement, they might have multiple therapeutic7effects in arthritis.
In a mouse model of systemic lupus erythematosus, MSCs
were able to reconstruct the osteoblastic niche, and to restore
immune homeostasis, conferring significant therapeutic effect[40]. In a clinical study, patients showed improvement in serologic markers and renal function [44]. However, it appears thatin patients with systemic lupus erythematosus, the source of
MSCs has some importance. Autologous MSCs had no beneficial
effect on disease activity, but allogeneic bone marrow or cord
blood MSCs produced an improvement in clinical and laboratory parameters. This suggests that, at least in some forms ofsystemic lupus erythematosus, MSC functions could be impairedby the underlying disease.
In a chemically-induced model of autoimmune colitis, thesymptoms were significantly improved by systemic infusion ofMSCs, and clinical studies on patients with perianal fistulae due
to Crohn’s disease showed encouraging results. Administration of
MSCs has induced healing of inflammatory ulcers and improved
nutritional status [45]. So far, inflammatory bowel diseases
appear to be a responsive target for the immunomodulatory
effects of MSCs.
MSCs and other pathologies
MSCs derived from the early human embryo can betransformed into epidermal cells in vitro and in vivo [46]. Thisfinding has led to the question whether MSCs can be used inorder to accelerate wound healing. Bone marrow derived MSCs
differentiate into dermal tissue after subcutaneous implantation in immunocompromised mice. In a clinical study, patientswith treatment-refractory dermatopathies showed significant
improvement of non-healing wound areas after autologous
MSC transplantation [47]. In diabetic foot wounds, injectionsof autologous skin fibroblasts and MSCs into the wound edges
decreased wound size, while increasing the vascularity of thedermis [48].
MSCs implanted into devitalized muscle grafts have beenshown to support peripheral nerve regeneration, while MSCs
injected into patients with chronic spinal injury transdifferentiatedinto neural stem cells, improved their electrical and functional
symptoms [24].
In patients with metachromatic leukodystrophy and Hurler’s
syndrome, infused MSCs induced significant improvement innerve conduction velocities [49].
In a pig model, MSCs from the apical papilla of tooth cotransplanted with periodontal ligament stem cells were able
to generate a root periodontal complex which could support a
porcelain crown, leading to the recovery of tooth strength andappearance [24].
MSCs injected into infarcted rat myocardium engrafted andimproved cardiac function and structure, through myogenesis
and angiogenesis, while also enhancing the survival of existing myocytes. If the MSCs are combined with erythropoietin
treatment, there is improvement of capillary density, as well as
reduction of infarct size and fibrotic areas [50].
MSCs regenerated segmental femur defects in sheep and8other large animal models, restoring bone morphology [51,52].
In diabetes, MSCs may induce the regeneration or the
proliferation of resident insulin-producing cells, contributing to
the repair of damaged islets, while also aiding in the long-term
complications of diabetes, such as neuropathy, nephropathy,
and cardiomyopathy [53]. They can also transdifferentiate intofunctional hepatocytes, inducing liver regeneration and offering potential cell replacement therapy in the treatment of liver
failure [54].
MSCs have also shown promise in the replacement and rebuilding of damaged structures, with the aid of three-dimensional(3-D) scaffolds made from biocompatible materials. The ideal
scaffold should be biocompatible and biodegradable, it should
mimic the extracellular matrix structurally and functionally,
while also offering a suitable environment for the growth anddifferentiation of MSCs.
The systemic inflammatory response syndrome (SIRS)
develops in response to severe sepsis, surgery, or trauma, due
to the dysregulated activation of the immune system. It causes
multiple organ dysfunctions, with a mortality rate of up to 40%[37]. In vitro studies have shown that MSCs can modify the immune response of SIRS [55]. Animal studies with MSCs havebeen performed, in models of endotoxemia [56], acute respiratory
distress syndrome (ARDS) [57], and intra-abdominal sepsis [58].
They showed a reduction of local and systemic inflammation,
improved hemodynamics and organ function, reduced organ
injury, and improved survival.
CONCLUSIONSThe current data suggest that MSC may constitute a
promising alternative strategy in the treatment of a wide rangeof immune-mediated diseases, through their immunomodulatory
effects, but also as systems of drug or gene delivery to diseased
tissues or organs.
They can prove to be useful in GVHD, autoimmune diseases, organ transplantation, neurodegenerative diseases,
cardiac failure, burns, bone defects, etc.
However, the clinical immunological outcome may notalways be predictable, because it depends on environmental
factors that cannot be reproduced in vitro.
Directions for future research should include:• Standardization and validation of MSC isolation andexpansion methods;• Animal studies to determine the underlying mechanismsof the MSC immunosuppressive effects;• In vivo tracking of MSCs in patients;• Multicenter randomized clinical trials, to further assess
MSC safety and efficacy.
ACKNOWLEDGMENTS
This work was supported by CNCSIS-UEFISCSU, project
number PN-II-IDEI-PCE 318/2011 and by the Sectorial Operational Programme for Human Resources Development, financed fromthe European Social Fund, FSE POSDRU/89/1.5/S/60746.
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INTERACTINEA DINTRE CELULELE STEM MEZENCHIMALE SI EFECTORIICELULARI AI SISTEMULUI IMUNREZUMAT

Celulele stem mezenchimale reprezintă un subpopulaţie rară de celule stem cu sediul în măduva hematogenă, unde interacţionează
cu celule stem hematopoietice, ghidându-le creşterea şi diferenţierea, şi unde servesc drept rezervă pentru diferite ţesuturi mezenchimale. Acestea prezintă şi proprietăţi imunosupresive remarcabile, înhibitând proliferarea şi funcţiile majorităţii populaţiilor de celule
imune. Acest raport oferă informaţii cu privire la cercetările actuale în ceea ce priveşte interacţiunile celulelor stem mezenchimale
cu celulele sistemului imunitar, posibilele mecanisme prin care se desfăşoară aceste interacţiuni, rezultatele utilizării in vivo, precumşi alte potenţiale implicaţii clinice, şi direcţiile viitoare de cercetare.
Cuvinte cheie: celule stem mezenchimale, sistem imunitar, imunosupresie, aplicaţii clinice

10

Fiziologia - Physiology  2012.22.1 (73)RELATIONSHIP BETWEEN SERUM LEVELS OF INTERLEUKIN-2
AND LIVER INJURY IN CHRONIC HEPATITIS C VIRUS INFECTION
BEFORE INTERFERON TREATMENTMANOLE COJOCARU 1, SIMONA ALEXANDRA IACOB 2, DORINA BANICĂ 3, EUGENIA
PANAITESCU 2, DIANA GABRIELA IACOB 2
1“ Titu Maiorescu“ University, Faculty of Medicine, Bucharest
2“ Carol Davila“ University of Medicine and Pharmacy, Bucharest3“ Marius Nasta“ Institute of Pneumology, Bucharest

ABSTRACT
Background: The pathogenetic role of immune-mediated mechanisms in chronic hepatitis C virus (HCV) infection has not yet been elucidated. Objectives: To investigate the immune response to HCV through expression of IL-2 in the serum of chronically HCV-infected patientscompared to normal controls and its association with histological inflammatory indicators. Materials and methods: Fifteen patients withchronic HCV infection (6 male, 9 female, mean age 47.35±10.78 years) and 14 healthy subjects (6 male, 8 female; mean age 35.00±15.45
years) were included in this study. The diagnosis of the patients with chronic HCV infection was established on the basis of clinical, laboratory, ultrasonographic and histopathologic findings. The healthy subjects had negative hepatitis serology, normal liver function tests andnormal ultrasonographic findings. Results: Serum levels of IL-2 were increased in HCV patients as compared to healthy controls (385±41.2
vs.126±15.4 pg/mL; p<0.001). There was correlation between serum IL-2 levels, histological activity index scores (r=0.746; p<0.001) andserum ALT levels (r=0.714; p<0.001). Conclusion: IL-2 is the main mediator of the inflammatory responses to chronic viral infection and in
our study serum level of IL-2 in HCV infected patients before interferon treatment was higher than healthy subjects. Serum IL-2 levels wereincreased due to chronic HCV infection, and the amount of increase corresponds to the degree of inflammation.
Key words: chronic hepatitis C, IL-2, physiopathology of chronic hepatitis C virus infection, T lymphocytes

INTRODUCTION
Hepatitis C virus (HCV) is a ubiquitous virus infection. It has
been estimated that about 3% of the world’s population havechronic HCV infection. It is a major cause of chronic liver disease.
Chronic hepatitis C has been a public health concern during thelast decade in most developed countries (1). Host immune mechanisms are involved in chronic infection. The reason for the hepatocellular injury in chronic HCV infection is still unknown. Several
studies have suggested that T-cell immunoregulatory cytokines
play a key role in both HCV viral persistence and in extent of liver
damage. Factors involved in the progression to liver disease in
HCV infected patients are not well characterized. Little is known
about the production of IL-2 in chronic HCV infection. The IL-2
production by T cells is part a complex network in which a discrete
alteration is capable of disrupting the whole system (2, 3).
In this study, we aimed to investigate the immune response
to HCV through expression of IL-2 in the serum of 15 chronically
HCV infected patients compared to normal controls and also its
association with inflammatory indicators.
MATERIAL AND METHODS
Fifteen patients (6 male, 9 female, mean age: 47.35±10.78
years) with chronic HCV infection and 14 healthy subjects (6male, 8 female; mean age: 35.00±15.45 years) were included inthis study. Patients were consecutively recruited from the“ Matei
Bals“ Institute Infectious Diseases Division from April 2010-October2010. All of the patients and controls provided written informed

consent. All of the patients underwent a complete medical andlaboratory evaluation and a liver biopsy. The inclusion criteria forthis study were as follows: a confirmed HCV infection, a positive
PCR for HCV-RNA, increased levels of alanine aminotransferase(ALT) and a liver biopsy confirming chronic HCV infection. Measurement of serum IL-2 was performed for the cases and controls
using ELISA (R&D Systems). Serum samples were tested twice forIL-2. Comparisons of proportions between the patients and controls were assessed by Fisher’s exact test. Continuous data werecompared by Mann-Whitney rank sum test. The Spearman’s rank
coefficient was used to assess correlation between the continuos
variable. The results were statistically analyzed by Student t test
and a p<0.05 (95% CI) was considered statistically significant.
RESULTS
The mean of alanine aminotransferase (ALT) levels wereincreased (the range was 63 to 160 IU/L). The majority of patients(80%) were infected with HCV genotype I and 86% had an HCV
RNA viral load greater than 850.000 IU/mL. An increased expression of IL-2 has been documented in the serum of chronically HCV
infected patients before interferon therapy (385±41.2 vs.126±15.4
pg/mL; p<0.001). The serum levels of IL-2 were higher in malecompared with female but the differences were not significant(p>0.05). The highest level of IL-2 was detected in patients lower
than 32 years old. However, these differences were not significant(p>0.05). There was correlation between serum IL-2 levels andReceived August 5th, 2011. Accepted November 20th, 2011. Address for correspondence: Manole Cojocaru, MD, PhD, „Titu Maiorescu“
University, Faculty of Medicine, Department of Physiology, Center for Rheumatic Diseases, Bucharest, Thomas Masaryk No.5 Street,
Sector 2, RO-020983, Bucharest, Romania, phone/fax: +4021.324.30.13, e-mail: manole. cojocaru@yahoo. com

2012.22.1 (73)  Fiziologia - Physiology

11histological activity index scores (r=0.746; p<0.001) and serum
ALT levels (r=0.714; p<0.001).

Further studies are warranted on the role of IL-2 in chronic
HCV infection.

DISCUSSION
Host immune response is important in determining theoutcome of chronic HCV infection. Results of our study showedelevated serum levels of ALT (4).
The immunopathogenesis of chronic hepatitis C virus (HCV)
infection is a matter of great controversy. It has been demonstrated that T cells play a role in HCV clearance in HCV infectedpatients. Helper T cells (Th) help in the functions of the immune
system as the major regulator and also help to destruct antigen
and to reinforce antibody production. We found that there was agreater expression of IL-2 compared to the controls. IL-2 reflects
the degree of histological and biochemical activity of chronic HCV
infection (5, 6).
Immune-mediated mechanisms are believed to play animportant pathogenetic role in HCV infection. It is thought thatcytotoxic T lymphocyte responses early in infection may be
important for viral clearance. Several cytokines and chemokines
induced by viral infection play directly or indirectly roles in antiviral
defense. Cytokines are key mediators of inflammation, apoptosis,
necrosis and fibrosis and they are actively involved in the regeneration process of liver tissue after injury. Th1 cytokines, suchas IL-2, interferon gamma (IFN-γ), and tumor necrosis factor-alfa(TNF-α) are required for host antiviral immune responses. IL-2 isconsidered to be a Th1 type cytokine and is involved in enhancing
the proliferation and activation of most T lymphocytes, NK cells
and B-lymphocytes. Liver sinusoidal and inflammatory cells havebeen reported to be sources of IL-2 and no consensus exists on
the predictive value of this cytokine (7). Apparently, expression of
IL-2 is associated with a more advanced stage of disease. There
was a correlation between ALT levels and IL-2 expression. The
level of expression of type 1 cytokines, such as IL-2 and IFN-γ,
is correlated with the degree of histologic injury. IL-2 levels may
contribute to the role of innate immunity in stimulating the adaptive
immune responses (8, 9).

CONCLUSION
IL-2 may exert a pro-inflammatory activity. Serum IL-2 levels
increase due to chronic HCV infection, and the amount of increase
corresponds to the degree of inflammation.
CONFLICT OF INTEREST
The authors have declared no conflicts of interest.
Acknowledgement
This paper was supported by CNCSIS-UEFISCSU, PNII
1165/2009.
REFERENCES

1. Alter MJ. Epidemiology of hepatitis C virus infection. World J Gastroenterol.2007; 13: 2436-41.
2. Gramenzi A, Andreone P, Loggi E, Foschi FG, Cursaro C, Margotti M,
Biselli M, Bernardi M. Cytokines profile of peripheral blood mononuclear
cells from patients with different outcomes of hepatitis C virus infection.
J Viral Hepat.2005; 12: 525-30.
3. Tilg H, Kaser A, Moschen AR. How to modulate inflammatory cytokines
in liver diseases. Liver Int.2006; 26: 1029-39.
4. Pereira HM, Cavalheiro NP, Tengan FM, et al. Patients with chronic
hepatitis and normal transaminases. Rev Ins Med Trop.2005; 47 (5):247-51.
5. Jacobson Brown PM, Neuman MG. Immunopathogenesis of hepatitis
C viral infection: Th1/Th2 responses and the role of cytokines. Clin
Biochem.2001; 34: 167-171.
6. Guyton AC, Hall JE. Textbook of Medical Physiology.11th ed. Philadelphia, Saunders, 2006; 447.
7. Sun QL, Ran W. Review of cytokine profiles in patients with hepatitis.
World J Gastroenterol.2004; 10 (12): 1709-15.
8. Birk RW, Gratchev A, Hakly N, et al. Alternative activation of antigenpresenting cells: concepts and clinical relevance. Hautarzt, 2001; 52:
193-200.
9. Gramenzi A, Andreone P, Loggi E, et al. Cytokine profile of peripheral
blood mononuclear cells from patients with different outcomes of hepatitis
C virus infection. J Viral Hepat.2005; 12: 525-530.

RELAŢIA DINTRE NIVELURILE SERICE ALE INTERLEUKINEI-2 ŞI DISTRUGEREAHEPATICĂ ÎN INFECŢIA CRONICĂ CU VIRUSUL HEPATITEI B
REZUMAT

Introducere: Rolul patogen al mecanismelor mediate imunologic în infecţia cronică cu virusul hepatitei C (HCV) nu a fost încă elucidat.
Obiective: Investigarea răspunsului imun faţă de HCV prin concentraţia IL-2 în serul pacienţilor infectaţi cronic cu HCV comparativ cu
subiecţi aparent normali şi asocierea cu indicatorii inflamaţiei la nivel tisular. Pacienţi şi metode: Cincisprezece pacienţi cu infecţie
cronică HCV (6 bărbaţi, 9 femei, vârsta medie 47, 35±10, 78 ani) and 14 subiecţi aparent sănătoşi (6 bărbaţi, 8 femei, vârsta medie
35, 00±15, 45 ani) au făcut parte din acest studiu. Diagnosticul pacienţilor cu infecţie cronică cu HCV a fost stabilit pe baza datelor
clinice, de laborator ultrasonografice şi histopatologice
Subiecţii din lotul martor au avut serologia negativă pentru hepatită, testele pentru funcţia ficatului normale, datele de la examenul
ultrasonografic normale. Rezultate: Nivelurile serice IL-2 erau crescute la pacienţii cu infecţie cronică HCV comparativ cu lotul martor(385±41, 2 vs.126±15, 4 pg/mL; p<0, 001). Există corelaţie între nivelurile serice IL-2 şi scorul de activitate tisulară (r=0, 746; p<0,001) şi nivelurile ALT în ser (r=0, 714; p<0, 001). Concluzie: IL-2 este un mediator important al răspunsului inflamator faţă de infecţia
cronică cu virus, iar nivelul seric IL-2 la pacienţi cu HCV înaintea tratamentului cu interferon a fost mai crescut comparativ cu lotul
martor. Nivelurile IL-2 în ser erau crescute datorită infecţiei cronice cu HCV şi au corespuns gradului de inflamaţie.
Cuvinte cheie: hepatita cronică C, IL-2, fiziopatologia infecţiei cronice cu virusul hepatitei C, limfocite T

12

Fiziologia - Physiology  2012.22.1 (73)THERAPEUTIC PARTICULARITIES IN ADULT PATIENTS WITH
THYROID DISEASE AND DIABETESADRIANA GHERBON, LAVINIA NOVEANU, GEORGETA MIHALAS
Department of Physiology, UMF „V. Babeş“, Timişoara

ABSTRACT
Diabetes mellitus and thyroid diseases are two endocrinopathies frequently in general population. Because insulin andthyroid hormones are involved in cellular metabolism, excess or deficiency of one of these determinates functional
disorders of another. The purpose of this study is to determine the main therapeutic particularities in adult patients withthyroid disease and diabetes. The study group was represented by 650 cases, aged 18-80 years. The gender distribution
was net in favor of women being represented by 588 women and 62 men. Was used clinical, radiological, biochemical,
hormonal and immunological parameters.
Keywords: diabetes, thyroid disease, and treatment

INTRODUCTION
The thyroid disorders are common in the general population; their prevalence increases with age. The hypothyroidism
is the most common thyroid disease in adults and older women.
Its origin is usually autoimmune presenting as atrophic primary
hypothyroidism or Hashimoto’s thyroiditis. It may be also secondary to radioactive iodine treatment or thyroid surgery. In
rare cases, occurs secondary hypothyroidism to hypothalamicor pituitary disease (17).
The clinical hypothyroidism should be treated throughhormone replacement therapy. The most used replacement
therapy is with L-thyroxin. The usual dose is 1.6 µg L-thyroxin/
kg. Often, in the case of mild thyroid failure, the initial dose may
be lower and it is adjusted according to TSH value at every 23
months. With the normalization of TSH levels and titrating Lthyroxin dose, TSH should be monitored annually. In the caseof aggravation of the thyroid failure is necessary to increase
the dose of thyroxin. The L-thyroxin therapy in subjects withdiabetes may exacerbate pre-existing coronary heart disease
by increasing myocardial contractility and heart rate. Therefore,
it is better to start with a low dose, around 25 µg/day, followed
by a slow growth, monthly, with 25 µg/day, monitoring of clinicalstatus and TSH level (17).

The subclinical hypothyroidism should be treated inpatients who present increased LDLc, which is worsened byhypothyroidism, which present the antithyroid peroxydase
antibodies (antiTPO AB) detectable in serum (which means apossible transition to clinical hypothyroidism) and in patients withclinical manifestations (17).
By contrast, the hyperthyroidism is less common, witha ratio female/male 9/1. The Graves’ disease is most common
and usually affects young adults. The toxic multinodular goiterusually occurs in older people (17).
The hyperthyroidism can cause adverse effects such as
worsening of glycemic control and coronary artery disease.
For this reason, it is indicated definitive treatment with iodine
radiotherapy, whenever is possible. There are no contraindications for antithyroid medication at the diabetic patients, but
the Graves-Basedow disease long-term remission is lessthan 40%. The patients with toxic multinodular goiter or withautonomous thyroid nodules should be treated definitive byiodine radiotherapy or by surgery (17).
The patients with diabetes mellitus (DM) have an increased
prevalence of thyroid disease compared with non-diabetic
population (17).
Diabetes type 1 is commonly associated with endocrine andsystemic diseases with autoimmune etiology of type: Graves-

Received August 5th, 2011. Accepted November 20th, 2011. Address for correspondence:������������������������������������������������
Adriana Gherbon, MD, PhD, Department of Physiology, „Victor Babes“ University of Medicine and Pharmacy Timisoara, Romania, Eftimie Murgu Square No.2A, 300041, Timisoara, phone/fax:+40256220479; e-mail: adrianagherbon@yahoo. com
2012.22.1 (73)  Fiziologia - Physiology

13Basedow disease, Hashimoto’s thyroiditis, Addison’s disease,celiac disease, pernicious anemia, myasthenia gravis, vitiligo,
etc. (3). In patients with type 1 diabetes, as treatment is indicateddiet and insulin in various doses and regimens.
A particular association of type 1 diabetes with hypo-or
hyperthyroidism is characteristic to polyglandular autoimmune syndrome. There are three polyglandular autoimmune
syndromes.
1. Polyglandular autoimmune syndrome type I (PAS-I) is
characterized by the triad: cutaneous-mucosal candidiasis,
hypoparathyroidism and Addison’s disease.
2. Polyglandular autoimmune syndrome type II (PAS - II) (11,2) is the most common immuno-endocrinopathies, characterized
by the appearance into the same person of two or more of thefollowing diseases: Addison’s disease, Graves-Basedow disease, thyroiditis of autoimmune etiology, type I diabetes, primary
hypogonadism, myasthenia gravis, and celiac disease.
3. Polyglandular autoimmune syndrome type III (PAS - III)(1) is a PAS II syndrome but without adrenocortical involvement. This syndrome is associated with the following diseases:
celiac disease, hypogonadism, myasthenia gravis, sarcoidosis,
Sjogren’s syndrome, rheumatoid arthritis, gastric cancer, malabsorption due by pancreatic exocrine deficiency, and can beclassified into three subcategories:-PAS III A - autoimmune thyroiditis with type 1 diabetes-PAS III B - autoimmune thyroiditis with pernicious anemia-PAS III C - autoimmune thyroiditis with vitiligo and/or alopecia and/or other autoimmune diseases
The key of the successful management in patients with
autoimmune endocrinopathies is to identify and treat autoimmunity before it cause significant morbidity and mortality. The
treatment of organ insufficiencies is identical whether they occur isolate or in the polyglandular autoimmune syndrome. The
replacement therapy remains the cornerstone of the clinicalmanagement (8).
Educating patients regarding the nature of the disease isoften critical for early recognition of associated autoimmune diseases, and as in any chronic disease, the psychosocial supportshould be evaluated individually (8). The genetic counseling is
also justified at affected family members who required carryingout specific tests (8).
Emergency identification should be performed in all patientswith polyglandular autoimmune syndrome, because use ofhigh doses of corticosteroids in acute stress usually prevents
adrenal crisis in the patients with Addison’s disease, in thosewith adrenal autoantibodies and increased risk of adrenal insufficiency. Some authors believe that exogenous supplement withglucocorticoids administered during acute stress is indicatedalso at asymptomatic individuals with biochemical changes inadrenocortical disease (8).
From all endocrine components of the polyglandular autoimmune syndrome, only diabetes has not a favorable prognosis
after hormonal replacement therapy and appropriate monitoring(8).
14

All patients with PAS I and PAS II require hormone replacement therapy, vitamins and minerals. As a rule, they can andthey are advised to continue normal activities, except for certain
professions incompatible with the disease (eg. airline pilot who
has type 1 diabetes).
Because most diseases have in common endocrine autoimmunity, and there are many genetic studies at animal models,the perspective is gene therapy, as the immunostimulatory or
immunomodulatory therapy.
A number of studies also indicate an increased prevalenceof thyroid disease in patients with type 2 diabetes; the hyperthyroidism is the most common disorder seen (3). In the caseof association of impaired glucose tolerance (IGT) and fasting
impaired glucose tolerance (IFG) with thyroid disease, they usually occur as a result of excess of thyroid hormones.
In patients with type 2 diabetes the treatment is diet, diet andoral antidiabetic medication, or if is necessary, diet and insulin.
In patients with IGT and IFG the treatment is diet.
MATERIAL AND METHOD
Investigated population
The study included adult subjects with diabetes mellitus,
which in time present thyroid disease, or adult subjects withthyroid disease that subsequently present diabetes mellitus.
The study group comprised 650 cases, aged 18-80 years.
Gender distribution was net in favor of women being represented
by 588 women and 62 men.
Methods of investigation
Methods of investigation were the clinical data-history,
present status, and imaging-thyroid ultrasound, biochemistrycarbohydrate metabolism parameters: fasting bloods glucose,urine glucose, glycosylated hemoglobin and thyroid hormones
investigations and some immunological parameters.
Glucose determination was performed by enzymatic
techniques with glucose oxidase. Were considered normal
fasting blood glucose between 70-110 mg%, diabetes mellitusfasting blood glucose values above 126 mg%, impaired glucose
tolerance-fasting blood glucose values between 110-126 mg%
and the oral glucose tolerance test (OGTT) at 2 h between
140-200 mg% and fasting impaired glucose tolerance-fastingblood glucose values between 110-126 mg% and OGTT at 2h under 140 mg%.
Determination of glycosylated hemoglobin (HbA1c) was
achieved through the DiaStat program for glycosylated hemoglobin HbA1c that measures the ratio of glycated hemoglobin tototal HbA.
Determination of serum levels of TSH, free fraction of serum
level of triiodothyronine (FT3), free fraction of thyroxin (FT4) were
ARCHITECT quantitative method, which is an immunologic
determination by chemiluminescence’s with small Chemilumnescent Micro particle Immunoassay (CMIA). The following values
were considered normal: TSH = 0.465 to 4.68 mIU/ml, FT3 = 3.69
-10.4 pmol/l, FT4 = 10 to 28.2 pmol/l.
Fiziologia - Physiology  2012.22.1 (73)Immunological parameters were represented by somemarkers of thyroid autoimmunity-antiTPO and antithyroglobulin(antiTG) antibodies (AB). To determine the serum titers of antiTPO AB AxSYM antiTPO kit was used, the method is enzyme
immunoassay with micro particles, Meia (Micro particle Enzyme
Immunoassay). It was considered normal: antiTPO AB <35IU/
ml. To determine the serum titers of antiTG AB AxSYM antiTG
kit was used, the method is enzyme immunoassay with micro
particles, Meia (Micro particle Enzyme Immunoassay). It wasconsidered normal: antiTG AB <55 IU/ml.
Thyroid ultrasound performed in all cases is a non-invasive
method of exploration that allows measurement of thyroid volume, thyroid study report with cervical anatomical structures andthyroid parenchyma changes.
RESULTS AND DISCUSSION
Adult group was subdivided in function of the type at glycemic balance in 4 subgroups (Figure 1):− group with type 1 diabetes represented by 60 cases(9.23%)− group with type 2 diabetes accounted for 290 cases(44.61%)− group with impaired glucose tolerance (IGT) accounted
for 183 cases (28.15%)− group with fasting impaired glucose tolerance (IFG)
accounted for 117 cases (18%)

Case prevalence

50%

44,61%

40%
28,15%

30%

18%

20%
10%

9,23%

0%
DM type 1

DM type 2

IGT

IFG

Fig.1. Distribution of cases by type of changes in glycemic balance

The prevalence of the glycemic type treatment is shownin Table I.
Table I. Distribution of cases by glycemic type treatment
Treatment DM type 1
typen%
InsulinOAD
Diet

DM type 2
n

%

IGT
n

%

IFG
n

%

60 10016
5.51
148 51.03
126 43.44 183 100 117
Legend: OAD – oral antidiabetic medication

100

In the case of adults with diabetes type 1, all patients weretreated with insulin, in different regimens. In the case of adults
with type 2 diabetes, a rate of 51.03% of patients were treated2012.22.1 (73)  Fiziologia - Physiology

with OAD, 43.44% with diet and 5.51% with insulin. In the caseof adults with IGT and IFG, all patients were treated with diet.
Diet is the crucial element in the treatment of diabetes. It is essential to all forms of disease and it is only indicated therapy insome patients. Over one third of patients with newly discovered
diabetes can initially be balanced with diet.
The objectives of the nutritional therapy in diabetes are thefollowing (7):− maintaining blood glucose levels as close to normal− normalization of lipid profile− ensuring adequate caloric intake to achieve and maintainan optimal weight for adults− preventing and treating acute complications of diabetes(hypoglycemia, diabetic ketoacidosis) and chronic complications(diabetic nephropathy, autonomic neuropathy, hypertension,
cardiovascular disease)− improved quality of life through proper nutrition
The treatment with OAD is individualized. At the beginningof therapy it is account of blood glucose level, body weight,
severity of clinical symptoms. In some situations it is necessary
to associate two or more OAD. Sometimes it is necessary thetherapy with insulin or the combination between insulin and
OAD. In the case of insulin therapy, intensive insulin therapy ispreferred, with 3 or 4 injections of insulin/day. At the choice ofthe regimen it must take account of blood glucose level, patientage and its life expectancy.
A study in France showed that blood glucose self-monitoringin patients with type 2 diabetes improve glycemic control andreducing appearance of complications (6). The treatment of adulthypothyroidism is thyroid hormone replacement therapy (HRT).
Substitution is made for life. The treatment will take into account: patient age, duration of untreated disease, cardiovascularcomplications, etc. The benefits of the treatment appear after 7
days and reached maximum after 4-6 weeks. In elderly patients,
in patients with ischemic heart disease it starts with ¼-½ of theusual dose, with incremental doses. The total or optimal replacement dose can be achieved after 2-3 months of therapy. The efficacy is measured by clinical and laboratory criteria. It is monitor
patients for symptoms of overdose, which include tachycardia,
palpitations, irritability, headache, tremors and possible seizures
angina. The clinical and laboratory reassessment is carried out
every 6 months (15). The therapy with thyroxin in patients withpresent antiTPO AB is more or less controversial in euthyroid or
subclinical hypothyroid patients. Several authors recommend theestablishment of replacement therapy in patients with antiTPO
AB and subclinical hypothyroidism when TSH values>10 mIU/
ml, aged over 45 years. It is showed that 24% of patients who
have HRT for 1 year, after discontinuation replacement therapy
had a euthyroid status undetermined period of time. Remission
could be explained by the disappearance of AB blockers of
TSH or AT receptor and secondary of discontinuation treatment
with cytokine, lithium, and amiodarone. The treatment depends
also by goiter size. The results of drug therapy can be evaluated with ultrasound, measuring at different time intervals the
thyroid volume.
15

At 90% of patients, after the therapy with thyroxin, the goiter
fell by 30% after a period of 6 months. Under TSH, the titers ofantithyroid antibodies became smaller. In the cases with present
antibodies it is necessary the annual thyroid investigation, thescreening of TSH in thyroid functionality evaluation is preferred(14). In the case of the hyperthyroidism, the main treatment isadministration of synthesis antithyroid drugs (SATD). They are
initially administered at doses of attack (6-8 tab/day, for 4-6
weeks), followed by the maintenance dose (approximately 1/3 ofthe attack dose). The duration of therapy is 2 years. If the treatment is correct, 20-40% of the cases have lasting remissions.
The criteria for the therapy efficacy are: clinical (thyrotoxicosis
remission, goiter reducing), hormones (thyroid hormone values
normalization) and immunological (disappearance of TSI fromplasma). The growth of goiter reflects an excess of TSH by
overdose. In this case we can associate thyroid hormones for
short periods of time. The surgical treatment consists in subtotal
thyroidectomy and it is applied to cases of Basedow disease
with high goiter that does not respond adequately to SATD, toxic
adenoma and hyperthyroid goiter.
Treatment with I131 is applied to persons over 40 years, thosewith recurrence after thyroidectomy and those in which surgery
is contraindicated. It is not applicable to those with large goiter.
The disadvantage of this treatment is iatrogenic hypothyroidism,
which affects about 70% of cases in the first 10 years after radiation treatment (5). The treatment of thyroid disease at patientswith type 1 diabetes is shown in Table II.
Table II. Distribution of cases by thyroid disease treatment in patients with
type 1 diabetes
Thyroid
diseaseTreatment type
None Suppression Inhibition
n % n
%
n
%
6
100

Surgeryn%-GravesBasedow
disease (n=6)
Euthyroid
1100
diffusegoiter (n=1)
Thyroid3100
differentiatedcarcinoma(n=3)
ACT (n=50)
10 20 40
80
Legend: ACT = autoimmune chronic thyroiditis--

In the case of the patients with Graves-Basedow disease,
all were treated with Thyrosol (inhibition treatment), with anappropriate response. In the case of the patients with thyroid
carcinoma, all patients were treated with thyroid hormone (suppression treatment), all cases were operated, treated with I131 andpresenting iatrogenic hypothyroidism. In the case of the patientswith euthyroid diffuse goiter, all cases have received suppression
treatment with thyroid hormones. In the case of the patients with
ACT, 20% of cases did not require any treatment, and 80% ofcases received replacement therapy with thyroid hormones.
The treatment of thyroid disease at patients with type 2
16diabetes is shown in Table III.
Table III. Distribution of cases by thyroid disease treatment in patients with
type 2 diabetes
Thyroid
diseaseNone

Treatment type
Suppres- Substitu- Inhibition Surgery
sion
tion
n
%
n
%
n
%
n %
57 95
3 5

n
%
GravesBasedow
disease(n=60)
Autonomous
thyroidadenoma(n = 3)
Iodineinducedhyperthyroidism(n = 4)
Euthyroid 28 22.4 87
diffusegoiter(n = 125)
Nodular3 42.85 4
euthyroidgoiter(n =7)
Differ10
entiatedthyroidcarcinoma(n=10)
ACT
15 19.48 62(n=77)
Sub acute 4 100
thyroiditis (n=4)

-

-

-

-

-

3 100

-

-

-

4

100

-

-

69.6

-

-

-

-

10

8

57.15

-

-

-

-

-

-

100

-

-

-

-

-

-

80.52

-

-

-

-

-

-

-

-

-

-

-

-

-

In the case of the patients with Graves-Basedow disease,93.34% were treated with Thyrosol (inhibition treatment). Amongthe cases under inhibition treatment, 11 (18.33%) were presented
postoperative recurrences.5% were associated with large goiter,with compressive phenomena; postoperative recurrences weretreated with SATD. In the case of the patients with autonomous
thyroid adenoma, all 3 cases required surgical treatment. In thecase of the patients with thyroid carcinoma, they have received
suppression treatment with thyroid hormones, all cases were operated, treated with I131 and presenting iatrogenic hypothyroidism.
In the case of the patients with euthyroid diffuse goiter, 22.4%
of cases did not require any treatment. A percentage of 69.6%
received suppression treatment with thyroid hormones and 8%
of cases requiring surgery due to the presence of large goiter,with the compression phenomena. In the case of the patients with
ACT, 19.48% of cases did not require any treatment, and 80.52%
of cases received replacement therapy with thyroid hormones.
In the case of the patients with iodine-induced hyperthyroidism,
all cases were treated with Thyrosol (inhibition treatment). In the
Fiziologia - Physiology  2012.22.1 (73)

case of the patients with sub acute thyroiditis, it was practicedclassical treatment: anti-inflammatory, and then small doses of T4.
In the case of the patients with nodular euthyroid goiter, 42.85%
of cases did not require any treatment and will be evaluatedperiodically (ultrasound evaluation - thyroid cyst). A percentage
of 57.15% of cases have received suppression treatment withthyroid hormones.
The treatment of thyroid disease at patients with IGT isshown in Table IV.
Table IV. Distribution of cases by thyroid disease treatment in patients with IGT
Thyroid
diseaseGravesBasedow
disease(n=43)
Autonomous
thyroidadenoma(n=3)
Euthyroid
diffusegoiter(n= 81)
Nodulareuthyroidgoiter(n=2)
Differentiatedthyroidcarcinoma(n=2)
ACT(n=52)

None
n
-

-

%
-

-

Treatment type
Suppres- Substitu- Inhibition Surgery
sion
tion
n %
n
% n
% n
%
40 93.02 3
6.97

-

-

-

-

-

-

3

100

10 12.34 61 75.3

-

-

-

-

10

12.34

1

-

-

-

-

-

-

50

1

50

-

-

2

100

-

-

-

-

-

-

4

7.7

48 92.3

-

-

-

-

-

-

In the case of the patients with Graves-Basedow disease,
93.02% were treated with Thyrosol (inhibition treatment). A
percentage of 6.97% requiring surgery, after treated withthe thyroid hormone replacement therapy. In the case of thepatients with autonomous thyroid adenoma, all cases received
surgical treatment. In the case of the patients with thyroid
carcinoma, all patients were treated with thyroid hormones(suppression treatment), all cases were operated, treatedwith I131 and presenting iatrogenic hypothyroidism. In the caseof the patients with euthyroid diffuse goiter, a percentage of
12.34% of cases did not require any treatment, a rate of 75.3%
cases received thyroid hormones (suppression therapy)
and 12.34% cases required surgery due to the presence ofa large goiter, with the phenomena of compression. In thecase of the patients with ACT, 7.7% of cases did not require
any treatment, and 92.3% of cases received thyroid hormone
replacement therapy.
The treatment of thyroid disease at patients with IFG isshown in Table V.
2012.22.1 (73)  Fiziologia - Physiology

Table V. Distribution of cases by thyroid disease treatment in patients with IFG
Thyroid
diseaseGravesBasedow
disease(n=38)
Autonomous
thyroidadenoma(n=2)
Euthyroid
diffusegoiter(n= 39)Nodulareuthyroidgoiter(n=2)
Differentiatedthyroidcarcinoma(n=6)
ACT(n=29)
Sub
acutethyroiditis (n=1)

n
-

%
-

Treatment type
Suppres- SubstiInhibition
sion
tution
n %
n %
n
%
38
100

-

-

-

None

n
-

%
-

-

-

-

-

2

100

4 10.25 30 76.92

-

-

-

-

5

12.82

2

100

-

-

-

-

-

-

-

-

-

-

6

100

-

-

-

-

-

-

7 24.13 22 75.87

-

-

-

-

-

-

1

-

-

-

-

-

-

100

-

-

Surgery-

In the case of the patients with Graves-Basedow disease,
all were treated with Thyrosol (inhibition treatment). In the caseof the patients with autonomous thyroid adenoma, all havebenefited from surgery. In the case of the patients with thyroid
carcinoma, all patients were treated with thyroid hormone (suppression treatment), all cases were operated, treated with I131 andpresenting iatrogenic hypothyroidism. In the case of the patientswith euthyroid diffuse goiter, 10.25% of cases did not require
any treatment. A percentage of 76.92% of cases have received
treatment with thyroid hormone (suppression treatment), and12.82% of cases requiring surgery due to the presence of large
goiter, with the phenomena of compression. In the case of thepatients with ACT, a rate of 24.13% of cases did not require
any treatment, and 75.87% of cases received thyroid hormone
replacement therapy. In the case of the patients with sub acutethyroiditis, all patients did not require any treatment. In thecase of the patients with nodular euthyroid goiter (thyroid cyst),
all cases did not require any treatment and will be evaluatedperiodically.
If the thyroid disease was the first, excess thyroid hormone
or administrations of the thyroid hormone led to an imbalance ofpre-existing type 2 diabetes or unmasking latent one, and also
to appearance of IGT and IFG. Appeared type 2 diabetes was17easy type, requiring only treatment with diet.
If the diabetes preceded thyroid disease with hyperfunction,
its appearance has led to imbalance of the DM. Thus, the casesof type 2 diabetes treated with diet initially require treatment withOAD and who received OAD initially required a combination ofseveral OAD or insulin therapies.
In the case of the patients with type 1 diabetes, was a major
imbalance in 9 cases, manifested by diabetic ketoacidosis (3cases - mild, 4 cases - moderate and two cases - advanced).
The main thyroid disease that caused significant metabolic
imbalances in the type 1 diabetes case was Graves-Basedow
disease. Thus, insulin requirement increased from an averageof 0.65 U/kg to 1.3 U/kg in Graves-Basedow disease, at 0.9 U/
kg for thyroid carcinoma and to 0.78 U/kg for ACT.
A number of studies show the influence of thyroid diseaseon the metabolic balance. Thus, studies performed in Spain,
Japan and the U. S. stresses the role of thyrotoxicosis in thedevelopment of diabetic ketoacidosis, it is meeting in these
conditions not only in patients with type 1 diabetes but also in
those with type 2 diabetes (12, 10, 9, 4, 13). Also, studies in the
Czech Republic show the influence of the various endocrine
disorders association on metabolic control. The most severe
consequences are increased occurrence of hypoglycemia in
hypothyroidism and appearance of the diabetic ketoacidosis incase of thyrotoxicosis (16).
CONCLUSIONS
Thyrotoxicosis unmasks latent diabetes and worsens preexisting diabetes. In the patients with diabetes and hyperthyroidism,
the thyroid function returns to normal after antithyroid treatment,
which also improves glycemic balance.
The Graves-Basedow disease and diabetes is a particularpathogenic entity, the presence of uncompensated thyrotoxicosis
adversely affecting the evolution and prognosis of diabetes.
Because the Graves-Basedow disease presents a chronic evolution, it is relatively difficult to stabilized with antithyroid medication, especially its association with type 1 diabetes requires thecorrection of hyperthyroidism by thyroidectomy definitive (bysurgical or radiation).
The hypothyroidism can cause reduction of requirements forinsulin or oral antidiabetic medication in patients with diabetes.
The treatment with thyroid hormones in patients with diabetes
and hypothyroidism, improves the thyroid function, but worse
the metabolic balance. It also causes an increased prevalenceof dyslipidemia, aggravating arteriosclerosis.
In the case of the thyroid carcinoma, although its diagnosis
imposed the therapeutic maneuvers (total thyroidectomy, treatment with I131 and suppressive therapy with high doses of thyroid
hormones), that causes deterioration of glycemic balance, it isrecommended to continue the treatment with thyroid hormones
and to adapted diabetes treatment for prevent the occurrenceof local recurrence.
The diffuse and nodular euthyroid goiter imposed a suppression treatment with thyroxin to induce a subclinical hyperthyroid18

ism. In the case of a difficult control of diabetes it is required
thyroidectomy.
The polyglandular autoimmune syndrome including type1 diabetes, various thyroid diseases, CSR deficiency, ovarian
failure, severe systemic disease, worsen the glycemic balance,
caused mainly by associated medication: estroprogestative,
corticosteroids, immunosuppressive medication.
In the case of the patients with autoimmune endocrinopathies it is recommended annual determination of TSH, possibly
also of antithyroid antibodies in patients with type 1 diabetes(especially in the presence of significant family history) to capture
the onset of thyroid disease and to institute appropriate treatment. In the case of the pre-existing thyroid disease it is required
blood glucose monitoring to capture the onset of diabetes andto institute appropriate treatment.
From a therapeutic standpoint, the replacement therapy
remains the cornerstone of clinical management, the treatmentof organ deficiency are identical, whether they occur isolate orin the polyglandular autoimmune syndrome (PAS). Of all theendocrine components of the PAS, only DM has not a favorable prognosis even with hormone replacement therapy andappropriate monitoring. As adjunctive therapy it is required
the psychosocial counseling and genetic counseling, and ataffected family members would be useful to determine specificantibodies.
For perspective, the immunomodulating therapy remains;
this must be considered experimental and should be prescribed
only in controlled trials. Following the identification of new auto
antigens and better knowledge of the disease pathogenesis,
selective therapies will be introduced that does not cause generalized immunosuppression. Also, in the future it is provided
curative organ transplantation. Transplantation of pancreas orpancreatic islands is used in patients with diabetes and renal
impairment.
REFERENCES

1. Aung K, Salmon M. Polyglandular Autoimmune Syndrome, Type III.
In Endocrinology (electronic book), 2006, 1-18.
2. Betterle C, Lazzarotto F, Presotto F. Autoimmune polyglandular
syndrome Type 2: the tip of an iceberg? Clin Exp Immunol, 2004; 137(2): 225-33.
3. Cooper GS, Stroehla BC. The epidemiology of autoimmune diseases. Autoimmun Rev, 2003; 2 (3): 119-25.
4. Duun EH, Nygaard B, Nerup J. Thyrotoxicosis at the onset of diabetes mellitus. Report of five cases. Ugeskr Laeger, 1998; 160 (29):4327-30.
5. Grammaticos P, Vasiliou O, Papanastasiou E, Gerasimou G, Balaris
V, Alexandris R, Liaros G, Dedousi E. The clinical and laboratory findings in Graves’ multinodular type disease. Hell J Nucl. Med., 2006; 9(1): 53-9.
6. Guerci B, Drouin P, Grange V, Bougneres P, Fontaine P, Kerlan V,
Passa P, Thivolet Ch, Vialettes B, Charbonnel B. Self-monitoring ofblood glucose significantly improves metabolic control in patients with
type 2 diabetes mellitus: the Auto-Surveillance Intervention Active (ASIA)
study. Diabetes Metab, 2003; 29 (6): 587-94.
7. Ionescu-Tîrgovişte C, Radulian G, Turcu L. Tratamentul dietetic.
În: Tratat de diabet Paulescu, Editura Academiei Române, 2004:

Fiziologia - Physiology  2012.22.1 (73)

1211-20.
8. Kukreja A, Maclaren N. Autoimmune Endocrine Disorders. In PediatricEndocrinology (electronic book), 2002, cap.6
9. Kunishige M, Sekimoto E, Komatsu M, Bando Y, Uehara H, Iyumi
K. Thyrotoxicosis masked by diabetic ketoacidosis. Diabetes Care,
2001; 24: 171.
10. Saitoh T, Akamine N, Sakai T, Serizawa S, Tohno H, Horie T. Slowly
progressive insulin-dependent diabetes mellitus in an elderly patientwith Grave’disease. Intern Med, 2000; 39 (11): 943-6.
11. Sivarajah S, Fan CY, Akinsoto OPA. Polyglandular Autoimmune
Syndrome, Type II. In Endocrinology (electronic book), 2006.
12. Sola E, Morrilas C, Garzon S, Gomez-Balaguer M, HernandezMijares A. Association between diabetic ketoacidosis and thyrotoxicosis.

Acta Diabetologica, 2002; 39 (4): 235-37.
13. Unger J. Case Study: A 62 – Year – Old Man with“ Brittle“ Type 1
Diabetes. Clinical Diabetes, 2002; 20: 37-39.
14. Vanderpump MPJ, Tunbridge WMG, French JM. The incidenceof thyroid disorders in the community:a twenty-year follow up of the
Whickham Survey. Clin Endocrinol, 1995; 43: 55-68.
15. Vanderpump MP, Tunbrigge Wm. Epidemiology and prevention ofclinical and subclinical hypothyroidism. Thyroid, 2002; 12: 839-47.
16. Vondra K, Vrbikova J, Bendlova B, Dvorakova K, Sterzl I, Vondrova
M. Differences in type I diabetes mellitus of young adults with andwithout thyroid autoimmunity. Exp Clin Endocrinol Diabetes, 2005;
113 (7): 404-8.
17. Wu P. Thyroid disease and diabetes. Clinical Diabetes, 2000; 18: 1.

PARTICULARITATI TERAPEUTICE ALE PACIENTILOR ADULTI CU AFECTIUNITIROIDIENE SI DIABETREZUMAT
Diabetul zaharat şi afecţiunile tiroidiene reprezintă două endocrinopatii frecvent întâlnite în populaţie. Deoarece insulina şi hormonii
tiroidieni sunt implicaţi în metabolismul celular, excesul sau deficitul unuia determină modificări funcţionale ale celuilalt. Scopul acestuistudiu este de a determina principalele particularităţi terapeutice la pacienţii adulţi cu afecţiuni tiroidiene şi diabet zaharat.
Lotul de adulţi studiat a fost reprezentat de 650 cazuri, cu vîrste cuprinse între 18-80 ani. Distribuţia pe sexe a fost netă în favoarea femeilor, fiind reprezentată de 588 femei, şi de 62 bărbaţi. Au fost utilizaţi parametrii clinici, imagistici, biochimici, hormonali şi
imunologici.
Cuvinte cheie: diabet zaharat, afecţiuni tiroidiene, tratament

2012.22.1 (73)  Fiziologia - Physiology

19

PELOIDOTHERAPY MODULATES CORTISOL`S AND
THYROTROPHIN-STIMULATING HORMONE`S BALANCESURDU TRAIAN-VIRGILIU1, 2, ION ILEANA1, SURDU MONICA3
1
Ovidius University Constanta, Faculty of Medicine
2
Department of Physical Medicine and Rehabilitation, Emergency Clinical County Hospital of Oradea
3
Department of Neonatology, Emergency Clinical County Hospital of Constanta

ABSTRACTThe main aim of the study is to evaluate the dynamic of serum level of cortisol and thyrotrophin-stimulating hormone during mud
therapy. Blood samples were harvested as follows: before the first treatment, at 24 hours after the first mud application and at theend of the balneal treatment. According to requirements of evidence-based medicine were applied normality tests of distribution andhomogeneity of variance values and t-test for comparison of sampling.
For plasma levels of cortisol, the comparative analysis (t-test) of the groups pointed out that final value for cold application showsa statistically significant decrease with 31.27% (p=0.04<0.05). The TSH variations after cold mud ointment increases significantly
statistic from the baseline (p=0.03<0.05) with 36.8%. This results show that the cold mud application ask more adaptive reserve than
mud bath. Under the action of peloid therapy cortisols and TSH balance is improved.
Key words: peloid therapy, cortisol, thyrotrophin-stimulating hormone

INTRODUCTION„The couple“ human body - peloid can be compared to acybernetic system with automatic adjustment. Such a systemshows reverse connection mechanisms (feedback) for functional correction. Keystone of the neuroendocrine system is themutual morphological and functional relation of hypothalamicpituitary humoral control of hypophysis function, which secretes
hormones for“ subsequent“ glands. Stress conditions (including
and especially cold thermal stimulation) generates the“ information“ of the hypothalamus about the situation in the peripheryand the respond with increased hormone secretion addressedto the pituitary gland. This“ information“ modulates, among
other hormones, the pituitary hormone secretion (which in turn
adjust the cortisol) and the thyroid-stimulating hormone secretion (involved in modulating metabolic and energetically cells
functions) (1, 2).
In this summary presented context, it seems logical andimportant to investigate the plasma levels of cortisol and thyroidstimulating hormone comparing the contrasting cure with thethermo neutral cure.
So, the main objectiv of the study is to evaluate the dynamics
of serum level of cortisol and thyrotrophin-stimulating hormone
during mud therapy and the secondary objectiv is to compare
the two forms of peloidotherapy.

MATERIALS AND METHOD
The study was performed on 25 hospitalized patients in
Balneal and Rehabilitation Sanatorium from Techirghiol durring
the summer of 2010. The inclusion and exclusion criteria wereapplied.
Inclusion criteria in study plot:• rheumatic deseas by degenerativ type and posttraumaticsequels of limbs who had correct indications for balneal cure,apparently healthy based on clinical examination and laboratory tests
Exclusion criteria:• any contraindication for balneal cure
• chronic inflamatory deseases, heart failure and/or hightblood pressure, endocrine, neurological disorders;• skin lesions witch forbid balneal cure and adjunctive
procedures.
Study plot presentation. The average age of patientswas 50.20±8.75 yrs. Age average for the group with thermo
neutral application (MB) was 49.41±9.43yrs, and for the groupwith cold application (CO) was 49.36±8.98 yrs. The distributionof the patients by type of application is balanced: 12 patients(48%) performed cold mud applications and 13 patients (52%)
performed the neutral thermic applications (mud bath and saltbath). The distribution of the patients by age, sex, pathology

Received September 16th, 2011. Accepted October 25th, 2011. Address for correspondence: Surdu Traian, BP Hasdeu Street No.120, Constanta, Romania, phone:+40724554756, e-mail: traian@surdu. ro

20

Fiziologia - Physiology  2012.22.1 (73)Tabel I. Study batch presentation
Age
group
30 -39Sex
Pathology type
Comorbidities
Men Women Degen- Post trau- Present Absent
erative matic
1
1
0
2
0
2

40 -49

2

9

8

3

3

8

50-59

1

5

1

5

3

3

>60

2

4

6

0

4

2

Total

6

19

15

10

10

15

Most of the patients belongs to the age group 40-49 yrs,
patients in activity, and preoccupied with their health, and theless belong to age group 30-39 yrs, presenting post-traumatic
pathology and without further sufferings.
In the age group <60 yrs was not present any patient suffering post-traumatic conditions, and in the age group 30-39 yrs
was not present any patient suffering degenerative disease.
Treatment applied. Cold mud application is a therapeuticalcomplex composed of successive contrast hot-cold, witch isdeveloped in two phases: first phase for about 20 minutes includes sun exposure on the warm sand from the solarium/beach(heating), followed by the mud ointement (cold stimulation), andthe second phase involves for about 30 minutes the exposure to
sunlight in order to install a mild hyperthermia followed by a coldbath in the lake’s water. After several days of treatement patients
extend the two phases by introducing at least one additional
immersion into the lake, before and / or after mud ointment.
Patients are instructed that the correct method of application
requires a single mud ointment.
During the study, we followed carefully the correctness ofthe therapeutic application, being presents at the solarium everyday. Our presence at the solarium was required, because despiteof all the indications they received, patients are tempted, at leastin the second week, to apply the mud twice.
Samples procurement. Blood harvesting was performedin the morning, before the breakfast and before the start of thetreatment, in three moments of the cure:⇒ at the beginning of the treatment (noted „in“);⇒ at 24 hours after the first mud application (noted „24“);⇒ at the end of the balneal treatment (noted „fin“).
RESULTS
1. Serum level of cortisol during peloidotherapy
Normal value for plasma level of cortisol is between 5 - 20
mg/day. Results obtained were tabled for systematization andstatistical study (Table II). In accordance with the requirementsof evidence-based medicine were applied normality tests of
distribution and homogeneity of variance values which showed
2012.22.1 (73)  Fiziologia - Physiologythat obtained distribution of values are normal for all the investigated moments and homogenous in the groups. Comparative
analysis (t-test) of the groups pointed out that only the final value
for cold application shows a statistically significant difference(p=0.04<0.05).
Tabel II. Statistic presentation of plasma level cortisol values

Average plot
± DS
Average group
MB±DS
Average group
CO ±DS

24 hours after
Before thethe first
treatment
mud application
(in)
(24)

At the endof thebalneal treatment (fin)13.68±6.34

13.94±6.00

11.52±4.10

11.87±6.41

11.98±3.06

12.66±3.52

15.35±6.88

15.75±7.49

10.48±4.45

t-test p<0.05
MB/CO
MB in/MB 24,
fin
CO in/CO, 24,
fin

0.11

0.19

0.95

0.68

0.89

0.04

The values obtained for the whole group in dynamic, at thedetermination of plasma cortisol shows:• statistically insignificant slight increase immediately after
the first application• statistically insignificant slight decrease at the end of cure
(Figure1)

error bars mean +/- 1.0 SD

16.00
14.00
12.00

microg/dl

type and the presence/absence of the comorbidities are balanced (Table I).

10.00
8.00
6.00
4.00
2.00

13.68

13.94

11.52

cortizol in

cortizol 24

cortizol fin

0.00

Fig.1. Plasma cortisol values at the entire group

Graphic appearance is suggestive of the low level of variation, accounted for the entire lot:
The cortisol slight increase observed at the whole study
batch in the first twenty-four hours respectively after the firsttherapeutic application is the immediate adaptive response to
new conditions of stress generated by pain, travel, new surroundings, and different climate parameters from those in the area oforigin, medical consultation, and therapeutic application (2).
The comparative analysis of the cortisol levels by the type of
peloid therapy (Figure 2) shows major differences between them:• cold application produces intense stimulation• thermoneutral application is mild
21

microg/dl

Tabel III. Plasma TSH values and statistic analysis
18.00
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00

Before thetreatment(in)error bars mean +/- 1.0 DS

11.87

15.35

cortizol in

11.98

15.75

cortizol 24
M ud Bath

12.66

10.48

cortizol fin

Cold Ointment

Average plot
± DS
Average group
MB±DS
Average group
CO±DS

2. TSH variation on patients undergoing peloid
therapy
The metabolic response of local and systemic effects ofpeloid therapy is endocrine coordinated mainly by the adrenal
glands and thyroid (2). Normal value for TSH is 0.27-4.2 UI/ml.
In order to apply criteria for evidence based all values obtained
were recorded in tables (Table III). Statistical analysis of obtained
values shows that the distribution of values is normal for all
investigated times and homogeneous in the groups.
22

1.98±1.72

2.32±1.15

3.14±1.79

2.20±2.20

2.65±1.26

3.39±2.22

1.78±1.17

2.02±0.98

2.90±1.31

t-test (p<0.05)

Fig.2. Variation of plasma cortisol during different type of peloid therapy

MB/COMB in/MB24, fin
CO in/CO,24, fin

0.18

0.52

0.55

0.20

0.58

0.03

Comparative analysis (t-test) of the groups pointed outthat for the cold mud ointment the values obtained at the end of
the application differ significantly from the baseline and thoseobtained from application thermo neutral (p=0.03<0.05). The
aspect is suggestive of how reacts the secretion of hormone at
the adaptive needs of the body on balneal cure: the growth ofmetabolic needs as the thermoregulatory causes a progressive
increase of the thyrotrophin-stimulating hormone till the end of
the cure (Figure 3).

error bars mean +/- 1.0 SD

3.50
3.00
2.50

UI/ml

Cold application is a contrasting thermal stres request(thermal contrast is performed 4-5 times during the 1.5 - 2hours how long the application is) and that add to the otherstressed factors of the balneal cure (3). Daily stimulation leads
to increased consumption of plasma cortisol during the first daysof treatment, which require adaptation of the secretory system.
This is illustrated by the slight growth of plasmatic level of cortisolin the determination made at twenty -four hours after the first
application. Add to this the fact that under the combined action ofheliotherapy, the immersion in the lake’s water and other therapeutically applications (massage, electrotherapy, physiotherapy)
possible subclinical joint inflammations, rheumatic pain, andmany other factors are reduced or even removed in the second
part of the diet and thus secretory demand is lower.
Under the action of contrasting complex the thermoregulatory function is optimized in the“ hypothalamic thermostat“ level
and adapted secretory with prompt responses, so endocrine
secretory activity is reduced at the end of the cure. It should
also consider optimizing the sweat secretion at the end of thecure, which also means more efficient energy consumption,
including glucose.
Under the influence of heat contrast and information fromthermal receptors the function of the variable thermal effect isimproved in the peripheral thermoregulation zone by modulating
local flow, segmentation, and finally general and optimizing therelease of autocoids factors.
In addition to contrasting thermal therapeutic application
should be noted that the summer climate parameters are disruptive for the functioning of the body: high temperature and highhumidity of air increases energy consumption due to a hard tostabilize balance between breeze (favorable evaporation ofsweat factor) and high humidity (unfavorable evaporation ofsweat factor).

At 24 hours after
At the end
the first mud apof theplication (24)balneal treatment (fin)

2.00
1.50
1.00
0.50

1.98

2.32

3.14

TSH inTSH 24

TSH fin

0.00

Fig.3. Plasma TSH variation during peloid therapy

TSH variation during cold application shows the favorable
effect of cold ointment on thermoregulatory function by reducing
thyrotrophin-stimulating hormone releasing (Figure 4). During thecold phase of the application (mud temperature 200 - 240C andimmersion in the lake’s water at approximately the same thermal
parameters) is necessary to maintain skin temperature almost
unchanging (3). The thermo neutral application does not requiresignificant thermoregulation, on the contrary is gentle, soothing(during application the bath temperature remains almost constant). Thermal comfort during thermo neutral bath stabilizes the
hypothalamic thermostat does not require major neuroendocrine
responses, but rather addresses to the physiological area whereare minimal responses. The thermoregulatory answer depends
on the“ error“ referred to the periphery from the hypothalamic
set-point. When the amount of“ error“ is reduced or the numberof“ errors“ produced in reduced time is low then the adaptive
response is reduced (1).
Fiziologia - Physiology  2012.22.1 (73)error bars mean +/- 1.0 S.D

4.00

UI/ml

3.50
3.00
2.50
2.00

cure due to diminution of pain, inflamation and accomodation
to new conditons.
So, during peloidotherapy consumption/need ratio of cortisol appear to be rebalanced. Our results are similar with otherreferences on mud of different type.

1.50
1.00
0.50
0.00

2.20

2.65

1.78

TSH in2.02

TSH 24
M ud bath

3.39

2.90

TSH fin

Cold ointment

Fig.4. Plasma TSH variation during different type of peloid therapy

The final results at the end of contrasting balneal cure areconsistent with other studies performed under the same conditions but with other types of mud: respectively the reduction of
the plasmatic cortisol level.
Analysis of data from the application of cold mud treatment
highlights the contraindication of the contrasting cure in patientswith thyroid hyper function and those with thyroid at the limit of
the compensation because the risk of decompensating is major
owing to statistically significant increase in plasma levels of TSH.
Increase of serum level of TSH during thermo neutral applicationis statistically insignificant, and safety for the patient.
CONCLUSIONS
1. Cortisol level during peloidotherapy shows different
respose according to the type of application and to the intensityof thermal stimulation.
Thermoneutral application produces slight increase ofplasma level, statistic insignificant. The values obtained haveminimal differences, within physiological frame, showing that thetherapeutic application of mud is a safe procedure for patients.
Cold mud ointment produces statistically significant decrease of serum levels of cortisol with 31.27% at the end of the cure.
This results might show:• increased peripheral consumption of cortisol in the firstdays of cure produced by the hight level of stres;• decreased needs of cortisol în the second part of the

2. Variation of serum level of TSH is concordant with cortisol
evolution.
Thermoneutral application generates minimal differencesconcordant with the low level of thermal stimulation. Cold mud
application produces statistic significant increase of thyroid
stimulating hormone with:• 11.89% after first cold mud application;• 36.8% at the end f balneal cure.
Increasing level of thyroid stimulating hormone is produced
by diminution of tiroxine and triiodotironine (negativ feedback)due to modulation of cells metabolic activity. Interaction between
cortisol and TSH is also, negativ feed-back type. Peloidotherpy
does not brokes this type of interaction, only modulates it. So,
serum level of cortisol and TSH varies according with the typeof thermal stimuation. Cold mud ointment is stronger than mudbath but both are safe procedure for patients.
Abbreviations: TSH - thyrotrophin-stimulating hormone;
MB – mud bath; CO – cold ointment with mud.
REFERENCES

1. Badiu G. Adaptologie – stress, umană. Ed. Fundaţiei Andrei Şaguna,Constanţa, 1999, ISBN: 973 – 9262-61-1; 58-61.
2. Pizzoferrato A, et al. Beta-endorphin and stress hormones in patientsaffected by osteoarthritis undergoing thermal mud therapy. Minerva
Med, 2000; 91 (10): 239-45.
3. Emweka CS, et al. Soft tissue Thermodinamics before, during andafter cold pack therapy. Med Sci Sports Exerc, 2002; 34 (1):45-50.
4. Antoni FA. Hypothalamic control of adrenocorticotropin secretion:
advances since the discovery of 41-residue corticotropin-releasing
factor. Endocr Rev., 1986; 7: 351.
5. Stewart LQ, Roper JA, Young W 3rd, et al. Pituitary-adrenal responseto acute and repeated mild restraint, forced swim and change in environment stress in arginine vasopressin receptor 1b knockout mice. J
Neuroendocrinol, 2008; 20:597.

PELOIDOTERAPIA MODULEAZA ECHILIBRUL CORTIZONIC SI AL TSH
REZUMAT

Scopul principal al acestui studiu a fost evaluarea nivelului seric al cortizolului si al TSH in timpul terapiei cu namol. Probele sanguine
au fost recoltate in diferite puncte cheie ale terapiei: inainte de prima aplicare, la 24 de ore de la prima aplicare a peloidoterapiei si la
finalul tratamentului balneal. In conformitate cu cerintele medicinei bazate pe dovezi, au fost aplicate teste statistice de normalizare
a distributiei si omogenizare a valorilor variantelor, precum si testul „t“ pereche pentru compararea probelor.
Pentru evidentierea nivelului de cortizol a fost efectuata analiza comparativa (testul“ t“ pereche) a grupurilor studiate, care a relevant
ca valorile finale pentru procedurile de aplicare la rece prezinta o scadere semnificativa din punct de vedere statistic cu 31, 27%(p=0, 04<0, 05). Variatia TSH dupa aplicarea procedurilor peloidoterapeutice la rece a prezentat o crestere semnificativa statistic
fata de control cu 36, 8% (p=0, 03<0, 05) with 36.8%. Aceste rezultate arata ca procedurile terapeutice de apicare a namolului larece solicita rezerva adaptativa a organismului intr-o masura mai mare decat baile de namol. Sub actiunea peloidoterapiei, echilibrul
cortizonic si al TSH au fost imbunatatite.
Cuvinte cheie: peloidoterapie, cortizon, TSH

2012.22.1 (73)  Fiziologia - Physiology

23

MYASTHENIA GRAVIS: CARDIAC AND PHARMACOLOGICALCONSIDERATIONSBOGDAN ISTRATE1, CRISANDA VILCIU2, MAARTEN HULSMANS3, CARMEN TATU4
1
Katholieke Universiteit Leuven, Belgium, Faculty of Medicine, Department of Cardiovascular Diseases
2
Univestity of Medicine and Pharmacy “Carol Davila” Bucharest, Romania
3
Katholieke Universiteit Leuven, Belgium, Faculty of Medicine, Department of Cardiovascular Diseases, Atherosclerosis and Metabolism
Unit
4
Department of Physiology, UMF “Victor Babes”, Timisoara, Romania

ABSTRACT

Myasthenia Gravis (M.G.) is an autoimmune neuromuscular disease caused by antibodies that react with the nicotinic acetylcholine
receptor (nAChR) in the postsynaptic region of the neuromuscular junction. It is already known that some M.G patients, have also
antibodies that bind in a cross-striational type to skeletal and heart muscle tissue (striational autoantibodies).This article summarizes
what is known about the cardiac involvement in myasthenia, starting from molecular characterization of striational autoantibody types,
as well as the clinical presentation via some pharmacological particularities of the main therapeutical agent used in this disease.
Key words: myasthenia, cardiac involvement, striational antibodies, structure, immunopathology, anti-cholinesterases

STRIATIONAL MUSCLE ANTIBODIES RELATED TO
MYASTHENIA GRAVIS. MOLECULAR CHARACTERISTICS
AND CLINICAL PRESENTATION
1. Titin
Also known as connectin, is a giant protein (3000 kDa), thatfunction as a molecular spring, being responsible for the passive
elasticity of muscle. Titin is also important in the contraction of
striated muscle. It connects the Z line to the M line in the skeletaland cardiac sarcomere. Molecules of Titin are arranged so thatthey allow mechanical stability and tension in the sarcomere.
According to Romi et al., 2005, the main immunogenic region of
Titin is called myasthenia gravis titin-30 (MGT-30), and is locatednear the junction of the A/I band.
Another immunogenic region is located between the N1 andN2 lines. The expression of homologous immunoglobulin domain
is different. For example 15 modules are expressed in cardiac
muscle, while in the skeletal muscle such modules could be up
to 68 (Lubke et al., 1998). In sera containing MGT-30 antibodies,
antibodies to the I-band are present. M.G. patients who exhibit
antibodies to the I-band epitopes could have also antibodies
attached to the main immunogenic region. Anti-Titin antibody
were first put in evidence in sera of myasthenic patients, where
first discovered by Aarli et al., in 1990.
Clinical features: anti-Titin antibodies are found in 20-40%

of all M.G. patients and in 60-80% in patients older than 60.A
percentage of 32% were found in patients with non-thymomatous
myasthenia, older than 50 years. Myopathic signs in thesepatients were detected via electromyography.
Immunopathology: T cell proliferative response to MGT-30;
capability to activate the complement pathway; association withsevere M.G. concomitant with myositis.
2. The Ryanodine Receptor
The ryanodine receptor (RyR) is a calcium-release channel
located in the sarcoplasmic reticulum. There are two forms of
RyR: skeletal (RyR1) and cardiac (RyR2). The molecular weightof this receptor is about 565 kDa, containing 5035 amino-acids(Romi et al., 2005). The ryanodine receptor is mainly expressedin striated muscle tissue, but is also found in epithelium andneurons. RyR antibodies can generate allosteric inhibition of
RyR function in vitro, by inhibiting Ca2+, release from the sarcoplasmic reticulum. From antigenic point of view, cardiac andskeletal muscle RyRs are not the same but, anti-RyR antibodies
in MG patients cross-react with both subtypes of the receptor(Mygland et al., 1994).
Clinical features: Found in 13-38% of all myasthenia gravis
patients. Mean onset at age 57; M : F = 1:1; more severe thananti-Titin antibodies; bulbar, respiratory and neck involvement;
myocarditis and/or myositis.

Received October 20th, 2011. Accepted November 3rd, 2011. Address for correspondence: Dr. Bogdan M. Istrate, Department of Cardiovascular Diseases, Division ofExperimental Cardiosurgery, Katholieke Universiteit of Leuven,Faculty of Medicine, Belgium, e-mail: bogdan_ellis@yahoo.com

24

Fiziologia - Physiology  2012.22.1 (73)Immunopathology: Complement activation; inhibiting therelease of Ca2+, from the sarcoplasmic reticulum; activation of
autoantibodies to the dihydropyridine receptor and the transient
receptor potential canonical type 3.
3. Voltage-gated K+ Channel
Voltage-gated K+ channels (VGKCs) have four transmembrane α-subunits that can combine as homo/hetero-tetramers.
Kv1.4 is an α-subunit, with a molecular weight of 73kDa, thatis mainly located in the brain, peripheral nerves, skeletal andheart muscles.
Clinical features: 12-15% of all MG patients; mean onset
at age 49 years; M : F = 1:1; more severe than anti-Titin; bulbar
involvement and myasthenic crisis; lethal arrhythmias; myocarditis and/or myositis.
Immunopathology: Different from neuronal VGKC; QT- prolongation on electrocardiogram.

scheme of myasthenia gravis, is used a pharmacological agent
with a longer onset than neostigmine: Mestinon.
Mestinon (pyridostigmine bromide tablets) is an orally active cholinesterase inhibitor. Chemically, pyridostigmine bromide
is 3-hydroxy-1-methylpyridinium bromide-dimethylcarbamate(Figure 1).

OTHER STRIATIONAL ANTIGENS AND ANTIBODIES

Fig.1. Structural formula of Mestinon

Antibodies to human myosin High titers in patients with MG and
Antibodies to anti-actomyosin thymoma

Cardiac unwanted effects and their impact on heart
functiona) Bradycardia: occurs due to stimulation of muscarinic
receptors in the sino-atrial node. Bradycardia is more common
in children and after increased or high doses of drug.
b) Prolonged block due to reduced plasma cholinesterase
activity. This can occur due to congenital acquired causes andproduction of atypical plasma cholinesterase. Duration of prolonged block varies from 30 minutes to several hours.
It has been reported (Rowland et al., 2007) that some acetylcholinesterase inhibitors are capable to exert severe peripheralcholinergic effects and afterwards serious cardiovascular complications. Parasympathetic activity dominates neural controlof heart rate and cardiac function. Moreover, increased level ofacetylcholine stimulates GABA-ergic and glycine-ergic inhibitory
receptors by acting on vagal neurotransmission, and their vagal
activity via muscarinic receptors is translated by a slow heart rate.
As we suggested before, the acetyl cholinesterase inhibitors
can induce sinus bradycardia, sino-atrial block and aggregate
pre-existing sinusal node disease and atrioventricular block.
Profound sinus bradycardia or bradycardia related to secondor third-degree heart block could have the potential to generateheart failure, syncope or seizures. Some adverse effects of anticholinesterases are illustrated in Table I as well.

Anti-Rapsyn antibodies are also found in serum samples ofpatients with MG, Lupus and Chronic Procainamide associatedmyopathy (Aigus et al., 1998). Striational autoantibodies are
rarely found in AChR antibody-negative MG patients.
CARDIAC CONSIDERATIONS
Although relatively common, cardiac manifestations inpatients with M.G. may take several forms. Among these, we
mention asymptomatic ECG changes to ventricular tachycardia,
myocarditis, heart failure and sudden death, cardiac fibrosis andrhythm disturbances.
Changes in cardiac function via electrocardiogram are already known. This could be a terminal notching of the QRS complex, higher frequency of QT-prolongation, right bundle branch
block, sinus tachycardia, inverted T-waves, ST-depression orpoor progression of R-waves.
Anyway, the significance of ECG in MG evaluation related
to cardiac involvement remains uncertain.
The effect of acetyl-cholinesterase inhibitors on cardiac
function
In 2008, Owe and Gilhus demonstrated a normalizing ofcardiac tissue velocity and strain after administration of acetylcholinesterase inhibitors, but previous, in 1992, Johannesen
found an increasing peak of diastolic filling rate. Sometimes therhythm disturbances may be caused by the main medication of
the patient and/or the mechanism of the disease itself. Anticholinesterase drugs (also known as acetyl cholinesterase inhibitor) areused to reverse the effects of non-depolarizing neuromuscular
blocking drugs. This drug increases the concentration of acetylcholine at the level of neuromuscular junction via an inhibitory
activity of acetyl cholinesterase. Frequently in the therapeutic
2012.22.1 (73)  Fiziologia - Physiology

Table I. Frequency of cardiovascular adverse effects for anti-cholinesterases drugs

Adverse effectFrequency(%)
Dizziness and syncope
1-10
Bradycardia, atrial arrhythmias, myo- 0.01-1
cardial infarction, angina, seizures
Sino-atrial and atrioventricular block 0.001-0.1
Shire Pharmaceuticals Ltd, 2005; Elisai Ltd, 2006; Novartis
Pharmaceuticals UK Ltd, 2007*25c) Other effects: increased intra-ocular pressure, muscle
pain, nausea, increased intra gastric pressure, vasospastic
effects.
tors
tion.

Biochemical features of acetyl cholinesterase inhibiMestinon. Absorption. Distribution. Metabolism and excre-

Absorption. Only about 40% of an oral dose of Mestinon isabsorbed, afterwards significant quantities being destroyed in
the gastrointestinal tract. The presence of permanently charged
quaternary ammonium groups confers higher water solubility.
Thus, pyridostigmine comprises a great absorption in the duodenum, but overall absorption is poor and variable.
Distribution. Mestinon is mainly distributed in the extracellular fluid. It doesn’t enter in the central nervous system. The
plasma area under the curve after 4 hours is relatively constant(6000 to 10000 nanogram/ml/minute). Pyridostigmine has beenreported to cross the placenta and to decrease fetal plasmacholinesterase activity after increased oral doses.
Metabolism and excretion. Mestinon is highly metabolized and 95% is excreted in the urine. The main metabolite is
3-hydroxi-N-methyl-pyridinium bromide. Neither pyridostigmine
nor its metabolites are protein bound. Excretion is by filtration or
by hepatic conversion to the glucuronide. Competition for renaltransport mechanism by tertiary amines can occur.
Biochemical effects of cortisone in heart muscle
As is known already, cortisone and non-selective non-steroidal anti-inflammatory drugs are widely used to control symptoms
in osteoarthritis, rheumatoid arthritis, cardiomyopathies and otherdiseases with severe inflammatory bursts. With regards to MG
cortisone is the principal therapeutic agent (corticotherapy) but
sometimes with undesirable side effects. However, the attention
should be paid to the effects on exacerbation of congestive heart
failure and arrhythmias due to hypopotassemia. All steroids
exhibit positive inotropic and also inhibitory effects on the myocardial Na+-K+-ATP-ase activity .Modifications in the corticosteroid
structure, particularly in the steroid nucleus itself, are able toinfluence the pharmacodynamic properties and corticosteroid
metabolism even in the cardiac muscle.
CONCLUSIONSIn order to have an accurate evaluation of heart involvement
in MG a correlation between laboratory findings and clinical cardiac function has to be made. In our point of view any myasthenic
patient should be cardiologically evaluated with available modalities and specific tests to detect or to exclude the coexistence ofa cardiovascular dysfunction/disease towards with MG.
The detection of striational antibodies can provide information regarding cellular and molecular alteration in the heart
muscle tissue, allowing in this way a new classification andmanagement of MG patients. Due to the fact that the anticholinesterases drugs are the main therapeutical tool in thisdisease, their effects and impact on central organs and theirfunction, should be from time to time evaluated.
REFERENCES

1. Romi F, Skeie GO, Gilhus NE, Aarli JA. Striational antibodies inmyasthenia gravis: reactivity and possible clinical significance. Archivesof Neurology 2005; 62(3):442-6.
2. Lubke E, Freiburg A, Skeie GO, et al. Striational autoantibodies inmyasthenia gravis patients recognize I-band titin epitopes. J Neuroimmunol. 1998; 81: 98-108.
3. Aarli JA, Stefansson K, Marton LSG, Wollmann RL. Patients withmyasthenia gravis and thymoma have in their sera IgG autoantibodies against titin. Clinical and Experimental Immunology. 1990; 82(2):284-8.
4. Mygland A, Tysnes OB, Matre R, Aarli JA, Gilhus NE. Anti-cardiac
ryanodine receptor antibodies in thymoma-associated myasthenia
gravis. Autoimmunity. 1994; 17(4): 327-31.
5. Agius MA, Zhu S, Kirvan CA, et al. Rapsyn antibodies in myasthenia
gravis. Ann N Y Acad Sci. 1998; 841: 516-21.
6. Owe JF, Gilhus NE. Myasthenia Gravis and the Heart. ACNR, 2008;
8(5): Nov/Dec.
7. Johannessen KA, Mygland A, Gilhus NE, Aarli J, Vik-Mo H. Leftventricular function in myasthenia gravis. Am J Cardiol 1992; 69(1):129-32.
8. Rowland JP, Rigby J, Adam CH, Rowland R. Cardiovascular monitoring with acetylcholinesterase inhibitors: a clinical protocol. Advances in
Psychiatric Treatment 2007; 13: 178-84.

MIASTENIA GRAVIS: CONSIDERATII CARDIACEREZUMAT

Miastenia Gravis este o boala neuromusculara autoimuna cauzata de anticorpii ce reactioneaza cu receptorul nicotinic pentru
acetilcolina (nAChR) din regiunea postsinaptica a jonctiunii neuromusculare. Este deja cunoscut faptul ca unii pacienti cu Miastenia
Gravis poseda atat anticorpi care reactioneaza incrucisat atat cu tesutul muscular scheletic cat si cu tesutul muscular cardiac (anticorpi striationali). Acest articol, prezinta pe scurt ce este cunoscut despre implicatiile cardiace in Miastenia Gravis, plecand de lacaracteristicile moleculare ale tipurilor de anticorpi striationali, precum si prezentarea clinica, prin unele particularitati farmacologiceale principalului agent terapeutic folosit in această afectiune.
Cuvinte cheie: miastenie, implicare cardiaca, anticorpi striationali, structura, imunopatologie, anticolinesterazic.

26

Fiziologia - Physiology  2012.22.1 (73)REVIEWLIVER AND IMMUNITYSUCIU MARIA1, 2, SCURTU ILEANA3, ARDELEAN AUREL1
1
Western University “Vasile Goldis” of Arad, Departament of Natural Sciences, Arad, Romania
2“Babeş-Bolyai” University, Faculty of Biology and Geology, Biology Department, Cluj-Napoca, Romania,
3“Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania

ABSTRACTThe immune system is a compilation of cells and molecules that silently guard the body’s well-being. Due to the fact that the liveris the largest organ in mammals and has a constant flow of all kind of substances, toxic or nutritious, viruses or microorganism, it
needs to have strong protection. Sometimes this can backfire and the protection can turn into self-aggression, when the liver suffersfrom a type of autoimmune condition. There are many factors that can lead to the many diseases that the liver can suffer from, butthere are also a few liver helpers at hand.
Keywords: liver, immunity, autoimmunity, Kupffer cells

INTRODUCTION
The liver’s function as an immune organ is beginning to beappreciated more and more. It has one of the largest resident
populations of macrophages, natural killer cells, and naturalkiller T cells, all of which are key components of the innate immune system. The liver is in a permanent contact with allogenic
antigens due to the fact that they penetrate the body troughfood. This requires a strict control of inflammatory responses
to different antigens (13). Autoimmune diseases of the liver are
considered rare, but in the last centuries they have become moreprevalent. They are the result of a variety of mechanisms thatusually have a genetic predisposition as a starting point, andsome environmental, nutrition or work-related factors. Reducing
exposure to xenobiotics and resolving digestive problems cannormalize and maintain a good and healthy immune function(10).This review will focus on recent knowledge concerning thehepatic immune system and the pathologies involving it.
In the present review, the literature search was made using
the following keywords: “liver immunity”, “liver autoimmunity”,“inflammation in liver”, and other terms that were needed tocomplete the subject. The articles were found in the on-line
libraries of Pubmed, Springer Publications, the official site of
NIH and CDC and Google Scholar.
The components and their role in liver immunity
In the liver, only up to 80% of all cells are hepatocytes. The
other 20% are constituted mostly of liver sinusoidal endothelial
cells, which represent about half of the non-hepatocytes. The
remaining half of non-hepatocytes is made up of cells belonging

to the adaptive immune system (T and B lymphocytes, naturalkiller cells and natural killer T cells), the innate immune system(Kupffer cells and eosinophils) and the humoral factors suchas the complement system and interferon. The innate immune
system is better represented because of the strong traffic offoreign substances (21, 31).
In the perisinusoidal Disse spaces, the adult liver contains
a number of pluripotent hematopoietic stem cells that will eventually produce all lineages of leukocytes and red blood cells.
Innate immune lymphocytes, natural killer cells and T cells arepresent in the liver in high numbers, unlike in other organs or inperipheral blood. In normal conditions all of these cells exist in thesinusoidal space and in the liver parenchyma. Kupffer cells staytightly attached to the sinusoidal endothelial cells, and naturalkiller cells come in contact with them modulating the immune
response to different harmful agents. This status is altered inmost liver conditions (30).
Neutrophils are a type of phagocytic granulocytes orpolymorphonuclear leukocytes that have a characteristic staining pattern. They derive from the myeloid stem cell and are themost numerous and important cellular component of the innate
immune system. They circulate throughout the body until they areneeded to act at sites of infection and inflammation. Cellular infiltration of activated neutrophils, which produce oxygen radicals
and secrete other toxic mediators, may increase the inflammatory
response, leading to cell injury and death (16, 33).
Kupffer cells are macrophages, and it is in their nature tocontinuously mature from circulating monocytes that leave thecirculation to migrate into tissues throughout the body. They

Received October 5th, 2011. Accepted November 20th, 2011. Address for correspondence: Suciu Maria, Regional Center of Immunology of Transplant, Emergency
County Hospital Timisoara, 10, Iosif Bulbuca Bv., Timisoara RO-300736, Timis, Romania, phone: +40265220479, e-mail: maria.suciu5@gmail.com

2012.22.1 (73)  Fiziologia - Physiology

27

have a few important roles: to phagocyte bacteria or cellulardebris, to present antigens to the T cells, to release signals
and to remove senescent blood cells from the circulation. To
fulfill these tasks these cells use scavenger receptors from thecysteine-rich superfamily, and produce interleukins IL-1, IL-6,
IL-12, tumor necrosis factor-α, transforming growth factor-β,
and reactive oxygen species. IL-12 activates the hepatic naturalkiller cells and natural killer T cells to produce interferon-γ, which
will activate the hepatic T cells. The result is phagocytosis andcytokine production by Kupffer cells. Besides being a foreign
body protection mechanism this is also an anti-tumor andanti-metastatic immunity process of the liver, delivered by theactivated natural killer cells and natural killer T cells located in theliver. Macrophages and neutrophils are the first line of defensein the liver (11, 14, 16, 28, 30, 31, 34).
Kupffer cells have the ability to eliminate various exogenousand endogenous substances. Recent experiments have shownthat Kupffer cells produce mediators that stimulate alcohol
metabolism. It was shown that the gut microflora-produced
endotoxin stimulates Kupffer cells to produce mediators, which
determine a hypermetabolic state of the parenchymal cells, followed by the development of hypoxia in the pericentral regionsof the lobule. Free radicals are produced with cell death as aresult (33).
Liver sinusoidal endothelial cells, although not immune
cells, have among their purposes to present antigens and tocreate strong bonds with Kupffer cells, leukocytes and platelets
in case of inflammation, a bond that will increase the concentration of signals, which in turn will help leukocytes migrate to theinjured hepatocytes (6, 34).
Natural killer cells derive from the common lymphoidprogenitor, develop in the bone marrow and can circulate in the
blood. They are larger than T and B lymphocytes, have distinctive cytoplasmic granules, and are functionally identified by theirability to kill certain lymphoid tumor cell lines in vitro without theneed for prior immunization or activation. The killing-mechanism
used by natural killer cells is the same as the one used by thecytotoxic T cells generated in an adaptive immune response.
Cytotoxic granules are released onto the surface of the boundtarget cell, and the effector proteins they contain penetrate thecell membrane and induce programmed cell death or apoptosis.
Natural killer cells are activated by interferon (IFN-α, and IFN-β)or macrophage-derived cytokines (IL-12). In their activated state
they are able to contain virus infections, and wait for the adaptive
immune response to produce the antigen-specific cytotoxic T
cells that can clear the infection. These cells present two typesof surface receptors for the cytotoxic activity: activating receptors
and inhibiting activation receptors, that won’t allow the killing of
normal, healthy cells. Even so, they lack the antigen-specificity
that is typical to the adaptive immune response. They have aprotective action against fibrosis, inhibiting liver fibrogenesis bykilling only the activated stellate cells which are known to mass
produce collagen in inflammation. They also induce apoptosis
or at least a metabolic freeze of the Ito cells using its production of interferon, thus having a liver protective status against
28

fibrogenesis (11, 16).
Hepatic natural killer T cells, also known as cytotoxic
T cells, are mainly used to kill viruses or intracellular bacteria
infected cells, which they recognize by the antigens displayed on
the surface of infected cells. Another role of natural killer T cells
is to release signals for Kupffer cells. Cytotoxic T cells typically
express the molecule CD8 on their cell surfaces that can recognize MHC class I molecules and store specialized cytotoxins,in granules, that are to be released when the infected target cellis met. The cells die by apoptosis induced by the holes createdin their cytoplasm by the killer cell, through which it introduces
its toxic content (16).
In liver injury cytotoxic T cells were found to balance thelocal production of pro-inflammatory (Th-1) and anti-inflammatory(Th-2) cytokines. The hepatic natural killer T cells themselves are
regulated by Kupffer cell produced cytokines, dietary factors, andcertain neurotransmitters, such as norepinephrine (21).
Ito cells, sinusoidal fat-storing cells or hepatic stellate cells
represent 30% of the liver immune cells and can be found in the
Disse space, between hepatocytes and sinusoidal endothelial
cells. They are not actual immune cells but act as such. A great
number of stellate cells inhibit T cell proliferation but will lose theinhibition control over B cell proliferation. They can store retinol
lipid droplets and regulate retinoid homeostasis. They become
myofibroblastic cells in liver injuries, lose the retinol accumulationand overproduce collagen. Ito cells are considered the primarycells to set fibrosis in the liver, and are related to alcoholic liver
steatosis and immune responses because they can interact withthe liver’s immune cells (11, 22, 31). Although it is thought that the
innate immune system unspecific recognizes a pathogenic factor,
it was demonstrated that it can very well recognize certain typesof proteins called pathogen-associated molecular patterns which
are located on the surface of the pathogen (e.g. lipopolysaccharides and peptidoglycans) using pattern recognition receptors(secreted, membrane-bound and phagocytic) (11).
Liver conditions
Autoimmune liver disorders are characterized by an inflammation of the liver, increased level and number of circulating
auto-antibodies and immunoglobulin G without a clear cause.
Autoimmune liver disorders include autoimmune hepatitis andautoimmune sclerosing cholangitis, chronic active hepatitis,
primary biliary cirrhosis (1, 24), but there are a few acquired
liver conditions that can, at some point, induce autoimmunity or
severe degradation of liver and can require a transplant. Such
conditions include the hepatic viruses and a syndrome of thesmall intestine.
Autoimmune hepatitis is characterized by mononuclear,plasma cells and eosinophils invasion in the periportal area.
The condition can grow into fibrosis, distortion of the hepatic
lobule, the formation of regenerative nodules and cirrhosis. It isa progressive, chronic disease that occurs both in children andadults, predominantly appearing in women (70% of patients),
and that’s cause is unknown. Blood analysis is characterized
by a high level of gamma-globulins and self-antibodies. The
Fiziologia - Physiology  2012.22.1 (73)

condition responds well to immunosuppressant medication andliver transplants have been successful in patients who haveno response to medication. There are two types of autoimmune hepatitis. Type 1 is the most common form, appearing inadolescence or young adulthood. Type 2 autoimmune hepatitis
occurs in 2 to 14 year old girls. Most of the time, autoimmune
hepatitis is associated with other immune disorders such as:
type 1 diabetes, proliferative glomerulonephritis, thyroiditis,
Graves’ disease, Sjögren’s syndrome, autoimmune anemia andulcerative colitis (3, 15, 19, 25).
Chronic active hepatitis affects hepatocytes and starts
with lymphocytes entering the parenchyma trough the portal areas and producing inflammation around the vessel. The presenceof toxins or viral and bacterial entities in the liver will determine
the leukocytes immune function to activate and the Kupffer cells
to produce IL-6 that will subsequently induce the production of
acute phase proteins and the complement system activation in
the hepatocytes (10, 30). The cause of autoimmune hepatitis is
unknown, but the illness presents the same characteristics as
any autoimmune liver disease: infiltration of leukocytes, highlevel of gamma-globulins and the presence in great numberof auto-antibodies. Women have an almost four times greater
prevalence than men (5, 15).
The hepatitis A virus is a picornavirus transmitted bycontact or ingestion of contaminated food or water. It is themost common hepatitis virus infection and it usually leads to a
total recovery in about three months. It passes undetected inchildhood, but when contracted in adulthood can cause severe
liver damage in individuals with autoimmune hepatitis. The two
conditions together lead to a rapid destruction of the liver, andthe presence of one can lead to the easy onset of the other (25,
27, 32, 35, 36).
The hepatitis B virus infection is a global health problem
affecting almost 400 million people worldwide. Many of theindividuals infected progress into chronic hepatitis leading tocirrhosis and tumors. In hepatitis B the blood CD56+ T cells
and natural killer cells do not decrease upon reaching the cirrhotic stage, but there are significant increases in CD8+ cells
and nonspecific cytotoxic T cells in patients with chronic severe
hepa­titis B (9, 26, 30, 32, 37).
The hepatitis C virus, belonging to the Flaviviridae family,
infects about 170 million people from all around the world, andin 80% of cases progresses to chronic liver disease (9, 38).
In hepatitis C, cirrhotic status natural killer CD56+ T cells andnatural killer CD56+ cells decrease until the majority are lost
(27, 30).
Hepatitis D is a contagious liver infection, which spreads
trough a virus that needs hepatitis B virus antigen, although it
is able to replicate independently of it. It is thought that someimmune-mediated mechanisms may be implicated in the progression of the liver damage. There are two types of hepatitis D
infections: hepatitis D coinfection, and hepatitis D super-infection.
The two differ in the amount of time passed from the momentof contacting the hepatitis B virus until contacting the hepatitis
D virus. The first one has a 5% chance of leading to a chronic
2012.22.1 (73)  Fiziologia - Physiology

infection, while the latter has an 80-90% chance. The condition
develops rapidly to cirrhosis or liver cancer (12, 27).
Primary biliary cirrhosis is a slowly progressive autoimmune disease of the liver that affects ten times more womenthan men, and peaks in prevalence around the age of fifty. It
is characterized by inflammation around the portal area and by
the immune controlled destruction of the hepatic bile ducts. It
is a distinctive condition by the fact that it affects mitochondria
within liver. Cirrhosis develops at about 10 to 30 years after the
onset of the disease (1, 10, 17, 29).
Primary sclerosing cholangitis is characterized byinflammation and fibrosis of both intrahepatic and extrahepatic
bile ducts, and is a progressive cholestatic disease that affects
children and young adults. It appears more often in men than
women, and peaks in prevalence around the age of forty. It manifests itself trough inflammation and destruction of the hepatic
bile ducts, both intrahepatic and extrahepatic. It is thought thatbacterial antigens are the starting point in genetically predisposed
patients. The activation of Kupffer cells leading to the overproduction of tumor necrosis may be connected to bile duct lesions.
The total number of circulating T cells in the body is decreased,
but is increased in the portal tracts. There are more CD4 thenCD8 lymphocytes in the circulation and an increased number of
B cells. Leukocytes cannot migrate because of the bile content,
but the T lymphocytes manifest an auto-reactivity towards thebiliary components and apparently epithelial cells act as autoantigens to lymphocytes. The condition gradually progresses tothe sclerosis of major bile ducts ending with cholestasis, fibrosis
and cirrhosis. It is usually associated with a few other immune
conditions: inflammatory bowel disease, celiac disease, diabetes mellitus, rheumatoid arthritis, Sjögren’s disease, systemicsclerosis, chronic pancreatitis, cystic fibrosis, retroperitoneal
fibrosis, sarcoidosis, thyroiditis, systemic lupus erythematosus,
lupus nephritis, autoimmune hemolytic anaemia, idiopathic
thrombocytopenic purpura or Langerhans cell histiocytosis. A
liver transplant remains the only alternative for patients in endstage primary sclerosing cholangitis (1, 2, 4, 20).
Intestinal Permeability, also called leaky gut syndrome,is a key condition to many autoimmune conditions. It has long
been recognized that untreated celiac disease, a primary causeof leaky gut, can lead to autoimmune hepatitis and other autoimmune conditions like insulin-dependent diabetes mellitus or
Hashimoto’s thyroiditis (10).
Kupffer cells, natural killer cells, natural killer T cells andCD8+CD122+ cells cooperatively act not only against bacterial
and viral infections, but also against cancers. Endo- or exotoxins
entering the liver circulation activate Kupffer cells and stimulatethem to produce IL-12. This interleukin determines natural killer
cells, natural killer T cells and CD8+CD122+ cells to generate
an antitumor activity. These cells can migrate to other organs
and inhibit a certain tumor growth there (30).
Possible causing agents
It was proven that estrogen treatment in vivo increases thesensitivity of Kupffer cells to endotoxins (33). Many agricultural
29

chemicals have been shown to have immune-modulating effects because of their estrogenic properties, and many othertypes of chemicals are now thought to induce, and possibly also
exacerbate, autoimmunity (10, 18). Cortisol maintained at a highlevel for a long period of time can determine adrenal depletion
and reduce its level. This will create violent immunoglobulin A
reactions followed by an autoimmune reaction (10).
Pesticides, fertilizers, and synthetic substances can trick
the immune system, and many of them can act as endocrine
disruptors because they mimic the estrogen form and function
and, as such, they affect every branch of the immune system:
innate cell-mediated and humoral immunity, and host resistance.
Vinyl chloride inhibits suppressor T cells and other immune
cells. Many other plasticizers and plastics, found in consumer
products such as food containers and wraps, are also known
as endocrine disruptors and are suspected of contributing toimmune dysfunctions (10)Females show a greater susceptibility to alcohol-induced
liver injury than males. Additionally, females who consume alcohol regularly and have been obese for 10 years or more are at
greater risk for both hepatitis and cirrhosis. Evidence has beenpresented that Kupffer cells are pivotal in the development of
alcohol-induced liver injury. It involves an increase in circulating
endotoxin, leading to the activation of Kupffer cells, which causes
an injury created by the difference between the presence and theabsence of oxygen in the tissue. Ethanol affects some Kupffer
cell functions such as phagocytosis, bactericidal activity andcytokine production. Serum tumor necrosis factor-alfa (TNF-a)
increases in alcoholics, supporting the idea that Kupffer cells
are activated in patients with alcoholic liver disease. TNF-α isproduced by Kupffer cell macrophages. During acute exposure
to ethanol, carbon uptake by the perfused liver increases about
25%, being a testimony that ethanol activates Kupffer cells (33).
Only 10 to 40% of heavy drinkers develop cirrhosis (31).
Fructose and lactose may overdrive a metabolic process
called glycation, which can normally appear when a small amountof these sugars is consumed. Glycation will alter the proteinsin such a way that the immune system will respond to them as
non-self and attack them. This process can be enhanced whenthese products are ingested from heated foods like brownedmeats and bakery products, especially those that are high infructose and lactose. Over-consumption of refined carbohydrates
leads to hyperglycemia, which also contributes to a high rate ofglycation that determines an overproduction of inflammatory
prostaglandins (10).
Anticonvulsants, beta-blockers, sulfonamides, estrogens,penicillin, and interferon have also been associated with autoimmunity (10).
Imbalances of gut bacteria can produce an immune response that triggers an inflammation of the mucosa which canlead to an autoimmune disease (7, 10, 11).
Liver support
In order to treat an inflammatory condition, the first step isto eliminate the cause. Because this is not possible for autoim30

mune diseases, the next thing to do is to apply a suppressionof the response to the antigens, self or non-self.
Autoimmune hepatitis responds well to corticosteroids while
primary biliary cirrhosis is manageable with ursodeoxycholic
acid. Primary sclerosing cholangitis has no clear treatment,
and usually ends in surgery. When nothing has worked andthe conditions have evolved to the later stages, there is often
a need for a liver transplant. For chronic hepatitis B and C, theviruses need to be inactivated by antiviral agents like nucleosideanalogues or interferon.
If foreign substances are ingested, the liver damage can be
minimized by removing that substance from the organism, as is
the case for drugs or alcohol (15).
Milk thistle or Silybum marianum, is a well known detoxifying agent, rich in flavonoids and antioxidants and especiallysilymarin, extracted solely for this purpose. Bupleurum (Bupleurum chinense; B. falcatum) is an anti-inflammatory, as well as a
stimulator of the production and release of bile, which facilitates
the detoxification process. Schizandra chinensis is considered
to have liver protective properties as it has the ability to normalize
liver enzyme levels (10).
CONCLUSION
It appears that from the last century the incidence of liver
disorders is getting higher by the year. Whether the causes areenvironment, the food we eat or unknown, these conditions area burden on the suffering and their family and friends. Therefore
there is a need for a clear big picture of the problem, in order toact specifically. Scientists may well work miracles and discovertreatments, but liver conditions will still be a constant problem
if we don’t change our lifestyles, in a way that won’t affect theenvironment, so that the environment won’t affect us.
Acknowledgments
This work was cofinanced from the European Social Fundthrough Sectoral Operational Programme Human Resources
Development 2007-2013, project number POSDRU/ CPP107/
DMI 1.5/S/77082, “Doctoral Scholarships for eco-economy andbio-economic complex training to ensure the food and feed safety
and security of anthropogenic ecosystems”
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FICATUL ŞI IMUNITATEAREZUMAT

Sistemul imunitar este o compilaţie de celule şi molecule care, întrun mod silenţios, păzesc bună-starea organismului. Deoarece
ficatul este cel mai mare organ la mamifere şi are un trafic continuu de substanţe diverse, fie toxice, fie hrănitoare, de virusuri saude microrganisme, el are nevoie de o bună protecţie imunitară. Acest lucru poate să fie uneori cu două tăişuri deoarece o protecţie
prea puternică poate deveni agresivă cu propriile structuri, fapt întâlnit în bolile autoimune ale ficatului. Există factori numeroşi carepot duce la diversele boli de care poate suferi ficatul, dar exită de asemenea şi câteva produse de ajutor la îndemână.
Cuvinte cheie: ficat, imunitate, autoimunitate, cellule Kupffer

2012.22.1 (73)  Fiziologia - Physiology

31

PREVALENCE OF CAROTID ARTERY STENOSIS AND MYOCARDIAL ISCHEMIA IN PATIENTS WITH PERIPHERAL
ARTERIAL DISEASEGRUICI A1, ARDELEANU E1, MIHĂESCU R2, MATUSZ AA3, ALAMAN R4, GURGUS
D1, GÂLCĂ E5
Family Medicine Department, “Victor Babes” University of Medicine and Pharmacy Timişoara, Romania
Semiology Department, Timis County Hospital3
Academic Society of Family Medicine, Timisoara, Romania
4
Cardiology Department, Cardiovascular Institute, Timisoara, Romania
5
Phisiology Department, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
1
2

ABSTRACTThe peripheral arterial disease (PAD), caused by localized atherosclerosis in the legs, is an important marker for generalized atherosclerosis and is closely related with CV events. The carotid arteries are among the vessels that are prone to develop atherosclerotic
lesions in the presence of risk factors such as cigarette smoking and hypertension.
Our aim was to evaluate the incidence and the risk factors mostly associated with the occurrence of carotid artery stenosis (CAS)and myocardial ischemia in individuals with PAD.
The study enrolled 648 patients, between 2007-2011, aged >50 years, mean age 63±5 years, 53% men and 47% women, fromfamily medicine offices of Timiş County. The median follow-up time was 34±6 months. The study inclusion criteria were definedas age between 50-79 years and the presence or history of PAD and at least one major risk factor for CV diseases. The ABI was
performed using a continuous wave Doppler probe and a sphygmomanometer. The PAD was defined as a resting ABI < 0.90 orwhen post exercise the ABI decreased at least in one leg with >20%. Only 151 patients matched with the study inclusion criteria
and were considered for carotid ultrasound and exercise stress test (EST), together with a similar control group. The carotid color
Doppler scanning was performed to detect and to quantify the hemodynamic severity of the CAS. An artery lumen stenosis >50%
was considered significant.
The values obtained from the ABI measurement ranged from 0.42 to 1.53, with a mean of 0.98. ABI <0.9 was found in 174 patients(27%). CAS > 50% was found in 26 patients (17%) of the study group and in 8 subjects (5.3%) of the control group (OR: 3.71; 95%
CI: 1.07-11.13, P = 0.031). The prevalence of CAS increased to 34% and 50% in patients who had three or four risk factors for CV
disease. EST was positive in 31 patients (20.5%) from the study group and in 17 (11.3%) control cases (OR: 2.03; 95% CI: 0.565.32, P=0.036). The incidence of CAS and positive EST was higher in elderly patients, smokers and in those with hypertension,
diabetes or dyslipidemia.
The ABI and the carotid ultrasound proved to be easy-to-perform, cheap and non-invasive investigations, with great importance inthe early diagnosis of the atherosclerosis elsewhere in the vascular system and with a significant contribution in predicting future
CV events in patients at high risk.
Key words: ankle-brachial index, carotid artery stenosis, peripheral arterial disease, myocardial ischemia, exercise stress test

BACKGROUND
The patients referred to the hospital or outpatient clinicwith symptomatic peripheral arterial disease (PAD) often havesymptomatic or asymptomatic manifestations of atherosclerosis
elsewhere in the vascular system, because atherosclerosis is a
generalized and progressive process (1).
The peripheral arterial disease, caused by localized atherosclerosis in the legs, is an important marker for generalized
atherosclerosis and is closely related with cardiovascular andcerebrovascular events (4). The incidence of fatal and nonfatal
cardiovascular events is two to three times higher in patientswith atherosclerosis in the lower limbs (5).
Although it has been shown that the PAD is a strong predictive factor for future cardiovascular events, previous studies

have revealed that only a small fraction of patients are early
diagnosed, most of the cases evolving asymptomatic (3,6). It isestimated that for every patient with symptomatic PAD, there areapproximately three up to four undiagnosed cases (2-7). Bothsymptomatic and silent PAD proved to have high levels of mortality and cardiovascular morbidity (3, 9). The risk of premature
death in a patient with PAD is three times higher than of the one
without peripheral vascular damage (5-8). Approximately 50%of the deaths are caused by myocardial ischemia, and 15%
are caused by stroke (4, 12). The presence of a co-existing
cerebro-cardiovascular disease increases mortality in patientswith PAD (10, 11).
The ankle-brachial index (ABI) is currently the most common
and useful diagnostic method, used to detect PAD, including

Received October 5th, 2011. Accepted November 20th, 2011. Address for correspondence Adrian Gruici, MD, PhD Student, Family Medicine Office, Family Medicine
Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania, phone: 0745149849, e-mail: adriangruici@yahoo.com

32

Fiziologia - Physiology  2012.22.1 (73)

asymptomatic cases (1, 2). This has come to a widespread useof the ABI measurement in the daily clinical practice and to theidentification of a high number of asymptomatic individuals with
PAD. In addition to its diagnostic value, the ABI can be usedin the evaluation of the generalized atherosclerosis (15, 16).
Previous studies have shown that an ABI<0.9 has a specificity> 98% for PAD and > 92% in the prediction of myocardial infarction and stroke (1).
The carotid arteries are among the vessels that are prone
to develop atherosclerotic lesions in the presence of risk factors
such as cigarette smoking and hypertension (18). It has beenshown that with high resolution two-dimensional ultrasound,the vessel wall characteristics of the carotid arteries can be assessed in an effective and accurate way at large-scale (19, 20).
This technique facilitates the evaluation of the lumen diameter,
the intima-media thickness and the presence and extent of theplaques of the carotid arteries (21).
The prevalence of coronary and cerebrovascular diseasein the asymptomatic population has not been investigated previously and for that reason, specific recommendations on thescreening for carotid stenosis or coronary artery disease cannot
be made at present.
OBJECTIVE
The aim of our study was to evaluate the incidence of carotid
artery stenosis (CAS) and of myocardial ischemia in individuals with PAD. We have also investigated which of the patient’s
characteristics were mostly associated with the occurrence of
CAS and myocardial ischemia.
MATERIAL AND METHODS
Study population
The study enrolled between 2007-2011 patients from 7 family
medicine offices of Timiş County. The average follow-up timewas 34±6 months.
The initial study population included 648 patients aged >50
years, who underwent a complete vascular screening at the timeof the enrolment to verify the study inclusion criteria and to study
atherosclerosis at the other sites of the vascular bed. They were
investigated by medical history, physical examination, vital signs,
ECG, laboratory analysis, echocardiography and noninvasive
markers for the screening of the atherosclerosis (intima-media
thickness and ankle-brachial index measurement). Written andinformed consent was obtained from all participants.
The study inclusion criteria were defined as age between
50-79 years, the presence or history of PAD and at least one
major risk factor for cardiovascular or cerebrovascular diseases.
Hypertension, diabetes mellitus, obesity, dyslipidemia and smoking were considered as CV risk factors.
The exclusion criteria were represented by age over 79
years, the patient refusal to participate in the study or who wereincapable of understanding the protocol, and those with problems
requiring hospitalization such as various forms of advanced cancer or other life-threatening diseases. The patients with stroke,2012.22.1 (73)  Fiziologia - Physiology

transient ischemic attack (TIA), carotid endarterectomy, atrial
fibrillation or those receiving treatment with drugs that could alter
the basal ST segment were also excluded from our study.
Cardiovascular Risk Factors and Events
The patients were evaluated in terms of cardiovascular risk
factors and cardiovascular events. The assessment was madeconsidering age, sex, smoking habits, alcohol intake, glucose
tolerance, systolic and diastolic blood pressure, total, and highand low-density lipoprotein cholesterol. Medical history included
data about current use of medication, alcohol intake, current andpast smoking behavior.
Physical examination included the measurement of height,
weight, abdominal circumference and body mass index (BMI). The
body mass index was calculated as weight to height squared. The
obesity was defined as BMI ≥ 30 kg/m2. The abdominal obesity
was defined according to the National Cholesterol Education
Program Adult Treatment Panel III (NCEP-ATP III) guide as
the abdominal circumference > 88 cm in women and > 102 cm
in men.
The blood pressure was measured at the right brachialartery using a random-zero sphygmomanometer in sitting andafter three minutes of standing position. The average of two
measurements, separated by a count of the pulse rate, obtained
at two different visits, was used in the study. Hypertension was
defined as a systolic blood pressure > 140 mmHg or a diastolicblood pressure > 90 mmHg or current use of antihypertensive
drugs for the indication of hypertension. Diabetes mellitus wasconsidered to be present when a subject was currently receiving
oral antidiabetic drugs or insulin treatment or had a fasting blood
glucose level > 126 mg/dl.
For the laboratory analysis we used a CardioChek-PA
analyzer with PTS-Panels test strips (provided by TerapiaRanbaxy). The blood samples were collected after an overnight
fast (fasting was defined as a self-reported interval of >12 hours
since the last intake of food) and were drawn for glucose, totalcholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides andketones. Dyslipidemia was defined as total plasma cholesterol> 200 mg/dl, triglycerides level > 150 mg/dl or the current use
of lipid-lowering medication.
The urinalysis was performed using Arkray AutionCheck II
equipment and Arkray AutionSticks EB5 urinary test strips (provided by Sanofi-Aventis). The urine samples were analyzed for
detection of glucose, albumin, creatinine, bilirubin, leukocytes,
erythrocytes, nitrites. For microalbuminuria these tests have fourlevels of interpretation: >10 mg/l (negative), >30 mg/l, >80 mg/l
>150 mg/l. In our study, microalbuminuria was defined as any
different interpretation of the negative. By using this equipment
we have also determined the albumin/creatinine ratio.
Signs and symptoms suggestive for peripheral arterial
disease include: intermittent claudication (pain of the lower limb
muscles appeared at walk and disappeared at rest), no pulse in
the leg arteries (pedal artery, posterior tibial artery, and popliteal
artery), ulcer or gangrene. The asymptomatic peripheral arterial
disease was defined as ABI ≤ 0.9 in at least one of the legs, but
33

in the absence of intermittent claudication.
Ankle-brachial index (ABI)
The presence of atherosclerosis in the lower limbs wasevaluated by measuring the systolic blood pressure (SBP) levelof the posterior tibial artery and the pedal artery at both sides
with an 8 MHz continuous-wave Doppler probe and a sphygmomanometer (Figure 1 a, b). The same test was performed at
the brachial arteries (1).
For the calculation of the ABI, we used the highest values
obtained (1). The ratio of the SBP at the ankle to the SBP at thearm was calculated for each leg. In patients with leg pain during
walking and having a normal resting ABI, we used an exercise
test to settle the diagnosis (1, 8). The peripheral arterial disease
was considered present when the resting ABI was <0.90 orwhen, post exercise, the ABI decreased at least in one leg with>20% (8, 11, 17).

a. Posterior tibial artery

b. Pedal artery

Fig.1. Technique for measuring blood pressure with Doppler probe

Ultrasonography of the carotid arteries
The carotid color Doppler scanning was performed to detectand to quantify the hemodynamic severity of the carotid arterystenosis. The carotid arterial scanning was made by a certified sonographist with a Sonoscape SSI-8000 high-resolution
ultrasonography system, equipped with a 10-Mhz linear arraytransducer. The transducer aperture was 46 mm.

head rotated away from the side being scanned (Figure 2 a, b).
The evaluation of the carotid plaques (mode B) was performedin longitudinal and transversal planes at the level of the common, bulb, and internal carotid artery. The presence of a plaque
was defined as a thickening of the arterial wall>1.5 mm (21).
Two measurements (longitudinal/ transversal) were made to
determine the grade of the stenosis (Figure 3 a, b).

a. Longitudinal section

b. Transversal section

Fig.3. Common carotid artery stenosis

In the stenosed area, the velocity of the flow is directly
proportional to the degree of the stenosis. The highest velocity
occurs at the level of stenosis or immediately downstream fromthe place of maximum narrowing. The severity of the stenosis
can be assessed by determining, at different times of the cardiac
revolution, the blood flow velocity and by calculating the ratios
between the determined velocities. An artery lumen stenosis of
more than 50% of the cross-sectional diameter was consideredsignificant, and was defined as a peak systolic velocity in theinternal carotid artery (ICA PSV) >125 cm/s, an end-diastolicvelocity (ICA EDV) >40 cm/s, and an ICA/CCA PSV ratio >2(Figure 4 a, b) (10).

a. Common carotid arteryb. Internal carotid artery

Fig.4. Carotid artery stenosis >50%

To demonstrate the association between PAD and myocardial ischemia, the patients underwent an exercise stress test
according to the modified Bruce protocol (7). A test was defined
as positive if there was a decrease in the ST segment of at least
1 mm measured at 80 ms from the J point.
Only patients with ABI <0.9 and one control group (onesubject for each patient, in ratio 1:1) were considered for statistical analysis.
Fig.2. Angles used for carotid artery scanning: a. transversal; b. longitudinal

The subjects were examined in a supine position with the34

Statistical analysis
The logistic regression analyses were performed to evaluate
Fiziologia - Physiology  2012.22.1 (73)the contribution of the low ABI and of each of the cardiovascularrisk factors (age, sex, hypertension, dyslipidemia, diabetes,
sedentary lifestyle) to the risk of carotid artery stenosis and of
an abnormal exercise stress test.
Differences between patients with and without peripheral arterial disease were represented by using the χ2 test and unpaired
t-student test. P<0.05 was considered statistically significant. All
the confidence intervals were calculated at a rate of 95%.
The calculations were performed using the statistical software package Statcalc Epi-Info Version 6.
RESULTS
The study enrolled, between 2007- 2011, 648 patients, aged
>50 years, from 7 family medicine offices of Timiş County. The
mean age of the study group was 63±5 years. A number of 346(53.4% ) were men and a number of 302 (46.6%) were women.
Baseline characteristics of the initial population are presented
in Table I.
Table I. Baseline characteristics of the initial population
Characteristic

Total(n=648)

ABI <0.9(n=174)

ABI >0.9(n=474)

P

Males (n, %)346 (53.4)

106 (61.2)

240 (51)

0.01

Age (years)

63±5

65±7

62±4

0.01

50-59

239 (36.9)

35 (20.3)

204 (43)60-69

260 (40.1)

73 (42)187 (39.5)

70-79

149 (23)66 (37.7)

83 (17.5)

Smokers (n, %)379 (58.5)

125 (71.8) 254 (53.6) <0.001

Hypertension(n, %)

403 (62.2)

121 (69.5) 282 (59.5)

0.02

Diabetes (n, %)221 (34.1)

75 (43.1)

146 (30.8)

0.01

BMI (kg/m2)

28.5

29.4

28.3

0.05

SBP (mmHg)

150

155

148

0.02

Age
groups(n, %)

Fig.5. Distribution of the study population according to the ABI values

In 9 patients, the carotid Doppler scanning could not be
performed because of logistic reasons and 14 patients did notgive their consent to continue the study.
In total, 151 (23.3%) patients were included for statisticalanalysis. 33 subjects (21.8%) were symptomatic, while in 118(78.2%) cases we found asymptomatic PAD (Figure 6). 94(62.2%) of them were males and 57 (37.8%) were females.

<0.001

Fig.6. Prevalence of symptomatic and asymptomatic PAD in the study group

DBP (mmHg)

87

89

87

0.1

LDL-C (mg/dl)

135

144

133

0.02

HDL-C (mg/dl)

44

39

45

0.02

In the study group, 48 (32%) patients had one risk factor,
55 (36%) had two risk factors, 36 (24%) had three risk factors
and a number of 12 (8%) had even four risk factors for cardiovascular disease.

Triglycerides(mg/dl)

148

157

145

0.01

Table II. Characteristics of the study group and control subjects

IMT (mm)

0.96

1.11

0.79

0.01

1

113 (17.4)

13 (7.5)

100 (21.1)

2

277 (42.7)

45 (25.8)232 (49)3

188 (29)

88 (50.6)

100 (21.1) <0.001

470 (11)28 (16.1)

42 (8.8)

No. of risk
factors (n,%)

Determination of the ankle-brachial index (ABI)
The values obtained from the ABI measurement ranged from
0.42 to 1.53, with a mean of 0.98. Normal ABI values, between
0.9-1.3, were recorded in 454 patients (70%). A number of 20
patients (3%) presented an ABI >1.3, 14 (70%) of whom werediabetics. An ABI <0.9 was found in 174 patients (27%), which
presented the study inclusion criteria and were considered
eligible for the study (Figure 5).
2012.22.1 (73)  Fiziologia - Physiology

Characteristic

Study
group
(n=151)

Controlgroup(n=151)P

Hypertension(n, %)

104 (68.9)

96 (63.5)

0.01

BMI (kg/m2)

29.4

29.2

SBP (mmHg)

155

144

DBP (mmHg)

89

87

LDL-C (mg/dl)

144

140

HDL-C (mg/dl)

39

41

Triglycerides(mg/dl)

157

153

ABI

0.81

1.11

<0.001

IMT (mm)

1.12

0.77

0.001

<0.00135

Ultrasound of the carotid arteries
Carotid artery plaques were found in 95 patients (63%)
compared to 56 patients (37%) of the control group (Figure 7).
26 patients (17%) of the study group presented a carotid arterystenosis (CAS) of 50% or higher on at least one side while inthe control group, a significant CAS was found in only 8 cases(5.3%) (Odds Ratio: 3.71; 95% CI: 1.07-11.13, P =0.031).

Table III. Characteristics of patients with and without CAS
Characteristics

Total
study
group
(n=151)CAS(n=26)No CAS(n=125)

P

Males (n, %)94 (62.2)

16 (61.5)

78 (62.4)-

Age (years)

63±5

69±4

60±6

0.001

Smokers (n, %)108 (71.5)

21 (80.7)

87 (69.6)

0.001

Hypertension (n, %)104 (68.9)

22 (84.6)

82 (65.6)

0.001

Diabetes (n, %)65 (43)

12 (46)53 (42.4)

0.05

BMI (kg/m )

29.4

27

29.8

-

SBP (mmHg)

155

160

153

0.05

2

Fig.7. Incidence of plaques and CAS
in the study and control groups

In the study group, 9 patients (34.5%) had a CAS of 70%
to 99% (2 of them bilateral), and 15 (58%) patients had a CASof 50% to 69% (3 of them bilateral) at least in one side. One
patient had an occlusion of the right internal carotid artery anda CAS of 50% to 69% on the left side, and another patient had
an occlusion on the right side and a stenosis less than 50% on
the left side (Figure 8).

DBP (mmHg)

89

85

89

-

Total Cholesterol(mg/dl)

215

245

199

0.001

LDL-C (mg/dl)

144

161

138

0.001

HDL-C (mg/dl)

39

37

40

0.01

Triglycerides (mg/dl)

157

167

153

0.001

Angina Pectoris(n, %)

27 (18)7 (27)

20 (16)0.001

IMT (mm)

1.12

1.26

1.07

<0.001

ABI

0.81

0.72

0.83

<0.001The prevalence of CAS increased from 17% (prevalencein all patients studied) to 34% (12 cases) and 50% (6 cases),
respectively, in patients who had three (24%-36 patients) or
four (8%-12 patients) risk factors (Table IV). The prevalence
decreased to 11% (6 cases) and 4% (2 cases), respectively,in patients with only two (36%-55 patients) or one risk factor(32%-48 patients).
Table IV. Prevalence of CAS in the study group according
to the number of risk factors

Fig.8. Types of CAS in the study group

The patients with CAS of 50% or higher were more frequently
women, older, active smokers, hypertensives, with higher levelsof total cholesterol, LDL-cholesterol and plasma triglycerides,
presenting angina pectoris and lower ABI (Table III).
36

No. ofrisk factors

No. ofpatients(n, %)

1
2

CAS (n, %)yesno

48 (32)2 (4)46 (96)55 (36)6 (11)

49 (89)3

36 (24)

12 (34)24 (66)4

12 (8)

6 (50)

6 (50)

Study
group

151

26 (17)125 (83)

Exercise stress test (EST)The EST was valid in 135 patients. In 16 subjects, there werebasal alterations in the ST segment or a left bundle branch block(8 cases), failure to reach sub-maximum heart rate (5 cases),or problems with the adaptation to the treadmill (3 cases). The
EST was positive in 31 (20.5%) patients from the study groupand in 17 (11.3%) control cases (OR: 2.03; 95% CI: 0.56-5.32,
P=0.036). The prevalence of a positive EST was higher in subjects with older age, hypertension, diabetes, and in those withhigh triglycerides or low HDL-cholesterol levels (Figure 9).
Fiziologia - Physiology  2012.22.1 (73)early detection of asymptomatic CAS is important to identify thepotential candidates for carotid endarterectomy.
The prevalence of a positive stress test was 20.5%. The
incidence of myocardial ischemia in the general population is
influenced by the techniques used in the evaluation, by the ageof the population and the presence of the cardiovascular risk
factors. In patients with diabetes, the presence of PAD is the best
predictive factor for coronary arterial disease. The subjects witha low ABI have a higher prevalence of segmental wall motion
abnormalities, as showed by echocardiography (16).

Fig.9. Prevalence of positive stress tests in the study and control groups

DISCUSSIONSThe peripheral arterial disease is widespread among theelderly, presenting an increased risk of major cardiovascular
events. In the general population, the incidence of PAD range
between 5 and 13% (16).In a population with atherosclerosis
and cardiovascular risk factors, the PAD range between 15%
and 40% (17).
The prevalence of PAD increased in elderly patients andin those with hypertension, diabetes or smoking habits (12). In
our study, the ABI confirmed the diagnosis of PAD in 27% of
patients, being very useful in detecting asymptomatic cases. The
association of multiple risk factors for atherosclerosis determined
an increased incidence of PAD.
Our study showed that the patients aged >60 years with a
low ABI and without known vascular disease had a prevalence of
CAS 3.7 times higher and of myocardial ischemia 2 times higher
than the subjects with a normal ABI. The CAS was present in
17% of patients, mostly in women, smokers, in elderly subjects,
in those with hypertension, and in those with elevated levels of
triglycerides, total cholesterol or LDL-cholesterol. Similarly, theprevalence of myocardial ischemia was higher in subjects withhypertension, diabetes, advanced age, high triglyceride or low
HDL-cholesterol levels.
The prevalence of CAS and of myocardial ischemia found in
our study may be used to estimate the efficiency of the screening for cardiovascular disease in this population. The results are
relevant given that the ABI is being increasingly used in primarycare health centers as well as in specialized outpatient clinics for
improving the stratification of the cardiovascular risk.
The incidence of CAS was higher in patients with three orfour cardiovascular risk factors: 34% and 50%, respectively. This
characteristic can be used to increase the chance of detecting a
CAS in patients with PAD but without known history of cerebrovascular disease (18, 20).
The search for CAS has been recommended in populations
in which the prevalence of significant stenosis (>50%) is >4.5%
and in which the annual rate of stroke is >3.3% per year (19,20). For this reason, several clinical guidelines recommend the
carotid scanning in patients with symptomatic PAD (19). The
2012.22.1 (73)  Fiziologia - Physiology

CONCLUSIONSIn conclusion, our study showed that in subjects aged
between 50 and 80 years and with at least one major risk factor
for cardiovascular disease, the presence of a low ABI <0.9 isassociated with a higher incidence of carotid artery stenosis (OR:3.71; 95% CI: 1.07-11.13, P =0.031) and of myocardial ischemia(OR: 2.03; 95% CI: 0.56-5.32, P=0.036) than in subjects witha normal ABI.
The prevalence of CAS increased to 34% and 50% in patients who had three or four risk factors for CV disease.
The ankle-brachial index and carotid artery ultrasoundproved to be easy-to-perform, cheap and non-invasive investigations, with a great importance in the early diagnosis of theatherosclerosis elsewhere in the vascular system and with asignificant contribution in predicting future cardiovascular andcerebrovascular events in patients at high risk. These parameters
can also improve the identification of patients at high risk.
Acknowledgement
This paper is supported by the Sectoral Operational
Programme Human Resources Development (SOP HRD)
2007-2013, financed from the European Social Fund andby the Romanian Government under the contract number
POSDRU/107/1.5/S/82839
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PREVALENŢA STENOZEI DE ARTERĂ CAROTIDĂ ŞI A ISCHEMIEIMIOCARDICE LA PACIENŢII CU BOALĂ ARTERIALĂ PERIFERICĂREZUMAT

Boala arteriala periferică (BAP), determinată de ateroscleroza localizată la nivelul membrelor inferioare este un important marker
pentru ateroscleroza generalizată şi este strâns asociată cu evenimente cardio- şi cerebrovasculare. Arterele carotide se regăsesc între
vasele predispuse a dezvolta leziuni aterosclerotice în prezenţa factorilor de risc, cum sunt hipertensiunea arterială sau fumatul.
Scopul lucrării a constat în evaluarea incidenţei şi a factorilor de risc asociaţi cu apariţia stenozei de arteră carotidă (SAC) şi aischemiei miocardice la persoanele cu BAP.
Studiul s-a desfăşurat între 2007-2011 şi a inclus 648 pacienţi, cu vârsta >50 ani, vârstă medie 63±5 ani, 53% bărbaţi şi 47% femei,
dispensarizaţi prin intermediul mai multor cabinete de MF din judeţul Timiş. Perioada medie de urmărire a fost de 34±6 luni. Criteriile
de includere în studiu au fost reprezentate de vârstă, cuprinsă între 50-79 ani, prezenţa BAP sau istoric de BAP şi cel puţin un factor
de risc major pentru boli CV. Acestora li s-a efectuat IGB folosind un dispozitiv Doppler, ei fiind supuşi şi unei evaluări detaliate dinpunct de vedere al factorilor de risc cardiovasculari şi al patologiei asociate. BAP a fost definită prin prezenţa IGB ≤0,9 în repaus.
Criteriile de includere în studiu au fost întrunite de 151 pacienţi care, împreună cu un lot similar de control, au fost examinaţi ecografic la
nivelul arterelor carotide şi au fost supuşi unui test de efort. Au fost considerate semnificative stenozele luminale mai mari de 50%.
Valorile obţinute la măsurarea IGB au variat între 0,42-1,53 cu o medie de 0,98. IGB <0.9 a fost obţinut la 174 pacienţi (27%). SAC>50% au fost întâlnite la 26 pacienţi (17%) din lotul de studiu şi la 8 indivizi (5,3%) din grupul de control (OR: 3.71; 95% CI: 1.0711.13, P =0.031). Incidenţa SAC a crescut până la 34% şi, respectiv 50%, la pacienţii care au asociat trei sau chiar patru factori derisc majori pentru boală CV. Testul de efort a fost pozitiv la 31 pacienţi (20,5%) din grupul de studiu şi la 17 indivizi (11,3%) din lotul
de control (OR: 2.03; 95% CI: 0.56-5.32, P=0.036). SAC şi ischemia miocardică au fost mai frecvent întâlnite la vârstnici, fumători,
diabetici şi hipertensivi.
IGB şi ecografia carotidiană s-au dovedit a fi investigaţii neinvazive, accesibile şi uşor de efectuat, având o mare importanţă în
diagnosticul precoce al aterosclerozei, cu o contribuţie semnificativă în predicţia evenimentelor CV viitoare la pacienţii cu risc înalt.
Cuvinte cheie: indice gleznă-braţ, stenoză de arteră carotidă, boală arterială periferică, ischemie miocardică, test de efort

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