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Fiziologia- Physiology 2011.21.2(70)
Fiziologia- Physiology 2010supplement

CONTENTS
1. Clinical Significance of Circulating Interleukin-8 Changes in Untreated Patients with Acute Psoriasis................................................................................................................. 4
Manole Cojocaru, Minerva Ghinescu, Inimioara Mihaela Cojocaru, Isabela Silosi, Sanda Marta Popescu
2. Hyperalgesia – An Overview .............................................................................................................................................................................................................................. 7
Daniela Motoc, Nicoleta Clarisa Turtoi, Francisc Schneider, Virgil Vasca, Elisabeta Vasca
3. REVIEW: Particularities of Association between Diabetes Mellitus and Thyroid Diseases................................................................................................................................... 11
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas
4. The Effects of Hypoxia on the Proliferation and Apoptosis of HUVEC Cultures in vitro........................................................................................................................................ 18
Cristina Maria Vaida Voevod, Ioana Baldea, Adriana Muresan
5. Expression and Significance of Ki-67 Antigen in Pulmonary Cancer. Relationship between Ki-67 and other Prognostic Factors........................................................................ 21
Mirela Grigoras, Elena Lazar, Alis Dema, Marioara Cornianu, Alexandra Faur, Laura S. Gotia
6. Detection of Anti-P80 Autoantibodies in Patients with Alzheimer’s Disease...................................................................................................................................................... 29
Manole Cojocaru, Inimioara Mihaela Cojocaru
7. Prevalence of High Risk Genotypes of Human Papilloma Virus among Fertile Women in the Western Part of Romania..................................................................................... 31
Zugravu Roxana, Gheorghiu Eleonora, Gheorghiu Georgeta, Cristea Anca, Hogea Elena, Dragomirescu Liliana, Licker Monica, Moldovan Roxana
8. Psychological Characteristics and Personality Traits Concerning the Adonis Complex........................................................................................................................................ 35
Diana Jivanescu, Iulia Crisan, Lazarescu M
9. Particularities of Lipid Metabolism Disorders in Patients with Diabetes Mellitus and Thyroid Disease............................................................................................................... 43
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas
10. Benefits of Thymectomy in Myasthenia Gravis................................................................................................................................................................................................ 49
Crisanda Vilciu, Bogdan Istrate, Carmen Tatu, Constanta Banica, Ana Campeanu,Constantin Popa
11. Experimental Neurophysiological Alterations Caused by Combined Nano-Manganese Exposure ................................................................................................................... 53
Zsuzsanna Máté, Andrea Szabó, Edit Paulik, András Papp

CUPRINS
1. Semnificatia clinica a modificarilor interleukinei-8 circulante la pacientii cu psoriasis acut netratat .................................................................................................................. 4
Manole Cojocaru, Minerva Ghinescu, Inimioara Mihaela Cojocaru, Isabela Silosi, Sanda Marta Popescu
2. Hiperalgezia – Privire de ansamblu..................................................................................................................................................................................................................... 7
Daniela Motoc, Nicoleta Clarisa Turtoi, Francisc Schneider, Virgil Vasca, Elisabeta Vasca
3. REVIEW: Particularitati ale asocierii dintre diabetul zaharat si afectiunile tiroidiene......................................................................................................................................... 11
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas
4. Efectele hipoxiei asupra proliferarii si apoptozei culturilor HUVEC in vitro......................................................................................................................................................... 18
Cristina Maria Vaida Voevod, Ioana Baldea, Adriana Muresan
5. Expresia si semnificatia antigenului Ki-67 in cancerele pulmonare. Relatia dintre Ki-67 si alti factori de prognostic......................................................................................... 21
Mirela Grigoras, Elena Lazar, Alis Dema, Marioara Cornianu, Alexandra Faur, Laura S. Gotia
6. Detectia autoanticorpilor anti-P80 la pacientii cu Alzeimer............................................................................................................................................................................... 29
Manole Cojocaru, Inimioara Mihaela Cojocaru
7. Prevalenta genotipurilor cu risc crescut a virusului Papilloma uman la femeile de varsta fertila, in partea de vest a Romaniei......................................................................... 31
Zugravu Roxana, Gheorghiu Eleonora, Gheorghiu Georgeta, Cristea Anca, Hogea Elena, Dragomirescu Liliana, Licker Monica, Moldovan Roxana
8. Caracteristici psihologice si trasaturi de personalitate in Complexul Adonis...................................................................................................................................................... 35
Diana Jivanescu, Iulia Crisan, Lazarescu M
9. Particularitatile modificarilor metabolismului lipidic la pacientii cu diabet zaharat si afectiuni tiroidiene........................................................................................................ 43
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas
10. Beneficiile timectomiei in Miastenia Gravis..................................................................................................................................................................................................... 49
Crisanda Vilciu, Bogdan Istrate, Carmen Tatu, Constanta Banica, Ana Campeanu,Constantin Popa
11. Alterari neurofiziologice experimentale determinate de expunerea combinata la nanoparticule si mangan.................................................................................................. 53
Zsuzsanna Máté, Andrea Szabó, Edit Paulik, András Papp
2011.21.2 (70)  Fiziologia - Physiology

3CLINICAL SIGNIFICANCE OF CIRCULATING INTERLEUKIN-8
CHANGES IN UNTREATED PATIENTS WITH ACUTE PSORIASISMANOLE COJOCARU1, MINERVA GHINESCU2, INIMIOARA MIHAELA COJOCARU3,
ISABELA SILOSI4, SANDA MARTA POPESCU5
1“Titu Maiorescu” University, Faculty of Medicine, Department of Physiology, Bucharest, Romania, 2“Titu Maiorescu” University, Facultyof Medicine, Department of Nursing, Bucharest, Romania, 3“Carol Davila” University of Medicine and Pharmacy, Department of Neurology, Bucharest, Romania, 4University of Medicine and Pharmacy, Faculty of Medicine, Department of Immunogy, Craiova, Romania,
5“Carol Davila” University of Medicine and Pharmacy, Department of Dermatology, Bucharest, Romania

ABSTRACT

Psoriasis in its various forms is one of the most common chronic inflammatory skin diseases worldwide. Interleukin-8 (IL-8) is a
pro-inflammatory cytokine. IL-8 is a potent chemoattractant for neutrophils, T cells and basophils.
The aim of this study was to compare the levels of IL-8 in adult patients with acute psoriasis prior to therapy and those of healthy
subjects and to investigate the relation of IL-8 levels to the severity of the disease. Serum levels of IL-8 were investigated in 39
untreated patients (28 women and 11 men, aged 33.76±4.2 years, age range 25-50) with acute psoriasis and 12 healthy subjects (8women and 4 men, aged 34.9±7.7 years, aged range 27-54). The production of IL-8 was measured by ELISA technique. Cytokine
concentrations (pg/mL) were expressed as the mean of duplicate samples. Results are given as the mean±SD. The mean valueof IL-8 of the patients group was 934±312 pg/mL (range from 629 pg/mL to 1246 pg/mL) and that of controls was 417±126 pg/mL(range 296 pg/mL to 539 pg/mL). Results were analyzed using a paired Student’s t-test. Wilcoxon’s rank-sum test was used as a
two-tailed test for statistical analysis of group differences. A p-value of less than 0.05 was considered significant. These analyses
demonstrated a significant increase in IL-8 production in acute psoriasis (p<0.01). No correlation was found between serum IL-8
levels and disease severity. Our data suggest that IL-8 may be of pathophysiological importance.
Key words: psoriasis, inflammation, serum IL-8

INTRODUCTION
Psoriasis in its various forms is one of the most common
chronic inflammatory skin diseases worldwide. It ranges frommild to severe, depending on how much of the skin is involved,
but in more severe cases almost the entire skin can be affected. Psoriasis affects both males and females equally and isindependent of race. There are two ages of onset. Early onset
disease (Type 1), which peaks at between 15 and 35 years ofage, is the most common and is often associated with a positive
family history. Late onset disease (Type 2) occurs at a peak ageof between 55 and 60 years. Although the precise aetiology ofpsoriasis is not known, its pattern (at least in the case of Type1 disease), fits that of an autoimmune disease. The onset oftype 1 disease occurs around the time of adolescence and its
appearance is gradual rather than sudden (1-4).
Interleukin-8 (IL-8) is a pro-inflammatory cytokine withchemoattractive and major activator properties on neutrophils.
The stimulation of T-cells involves activation signals from antigen
presenting cells (APCs). IL-8 is a cytokine with high specificity forneutrophils, on which it has chemoattractant and major activator properties. It may be produced by a large number of cells:
circulating monocytes, macrophages, endothelial cells and evenactivated neutrophils. The few studies in literature on the IL-8

behaviour refer to psoriasis (5-8).
This study proposed investigation of IL-8 behaviour in acutepsoriasis prior to therapy and to demonstrate the hypothesisthat IL-8 may have a pivotal role in determining the outcomeof disease.
MATERIAL AND METHODSIn order to provide a comparison with previously publishedresults in this study serum levels of IL-8 were investigated in
39 untreated patients (28 women and 11 men, aged 33.76±4.2
years, age range 25-50) with acute psoriasis and 12 healthy
subjects (8 women and 4 men, aged 34.9±7.7 years, aged range
27-54). Blood was harvested by venous puncture, in aseptic
conditions and with minimal venous compression at the timeof admission in hospital. The production of IL-8 was measured
by ELISA technique. Cytokine concentrations (pg/mL) were expressed as the mean of duplicate samples. Results are given as
the mean±SD. The IL-8 concentration was calculated based ona standard curve for each determination. Results were analyzed
using a paired Student’s t-test. Wilcoxon’s rank-sum test wasused as a two-tailed test for statistical analysis of group differences. A p-value of less than 0.05 was considered significant.
The project was approved by the Ethics Committee

Received March 10th, 2011. Accepted May 20th, 2011. Address for correspondence: Assoc. Prof. Manole Cojocaru, MD, PhD, “Titu Maiorescu” University, Faculty of
Medicine, Bucharest, 5 Thomas Masaryk Street 020983, Sector 2, Bucharest, Romania, e-mail: manole_cojocar@yahoo.com

4

Fiziologia - Physiology  2011.21.2 (70)RESULTS
Analyzing the behaviour of IL-8 in relation with acute psoriasis it is to be noted that increased serum IL-8 levels in 39 of thepatients with psoriasis prior to therapy as compared with thoseof healthy controls. The mean value of IL-8 of the patients group
was 934±312 pg/mL (range, 629 pg/mL to 1246 pg/mL) and thatof controls was 417±126 pg/mL (range 296 pg/mL to 539 pg/mL). These analyses demonstrated a significant increase in IL-8
production in acute psoriasis (p<0.01). No correlation was foundbetween serum IL-8 levels and disease severity. Our findingsthat IL-8 is increased in acute psoriasis could have pathogenetic
implications.
DISCUSSION
Our investigations demonstrate the major importance of IL-8
in psoriasis. The stimulation of T-cells involves activation signals
from antigen-presenting cells (APCs). These high IL-8 levels
in acute psoriasis point to inflammatory process in which IL-8,
a pro-infammatory cytokine may be involved. Our results correspond well with finding regarding high cutaneous IL-8 protein
expression, as determined by immunohistochemistry (7).
A publication has implied that production of IL-8 may beunder genetic control via a polymorphism at a functionaly active
site on the IL-8 promoter gene (9).
Among cytokines, TNF-α and IL-1 can induce IL-8 production. IL-8 sends signals through the neutrophil receptors boundto protein G. The signals received by the chemotactic receptorsof the neutrophils are essential for diapedesis and exocytosis of
the neutrophil granulations, rich in gelatinase, whose preferred
substrate is type IV collagen, a major component of the basal
membrane. T helper 2 (Th2) type responses are also characterized by production of IL-8. The local and systemic productionof cytokines is responsible for the majority of pathological andclinical consequences. IL-8 detection in the systemic circulationof the patients with acute psoriasis is essential for the investigation of the neutrophil involvement in the pathophysiology ofpsoriasis. Some evidence accumulated that epidermal cells areable to synthesize and release IL-8. These observations suggest that the IL-8 may be crucial in determining the degree ofT-cell proliferation. In addition to this local IL-8 overexpression,
endotoxin-stimulated whole blood from psoriatic patients showeda tendency for higher IL-8 formation capacity. IL-8 overexpression
may be a central phenomenon for the pathogenesis of psoriasis
and that its normalization would be beneficial (6,7,10).
Neutrophils have been much investigated, both because
this type of cell is a key factor in the inflammatory response andbecause the cytokines secretion may significantly influence thedirection and course of the disease. The few existing reports
on IL-8 in psoriasis are controversial. Chemotaxis, adhesion
or migrations of neutrophils in the vascular walls are essential
inflammatory phenomena that may be partially mediated by
IL-8. An interaction between T-cells and APCs is initiated whena specific antigen, presented in association with the MHC
on the APC, binds to T-cell antigen receptors. It is importantto caution that this is just one step in a very long process,2011.21.2 (70)  Fiziologia - Physiology

although it may be an important one. New treatments basedon T-cell pathophysiology have already shown considerable
promise (8,11-13).
Altogether, our study enforces earlier data that IL-8 is produced at local level to focus and amplify inflammatory responses.
Evidently, however, these local processes are not reflected by thesystemic IL-8 level even in cases of severely involved psoriaticskin. Consequently, serum IL-8 levels do not provide diagnostic
or prognostic criteria in psoriasis.
The neutrophil behaviour in patients with psoriasis constitutes the subject of current studies. Further investigations arenecessary to assess safety aspects as well as effects on diseaseactivity and related immunological parameters.
CONCLUSION
IL-8 overexpression may be a central phenomenon inpsoriasis. The present study provides further evidence that IL-8
deregulation may play a key role in psoriasis, may contribute toexacerbate psoriasis. The precise nature of IL-8 over-expression
is still unclear.
The IL-8 increase in acute psoriasis proves the involvementof neutrophils in the pathogenesis of the disease, IL-8 being oneof the most powerful chemotactic factors for neutrophils.
Acknowledgement
This work was made possible by a project grant from the
Romanian Academy of Medical Sciences.
REFERENCES
1. Schön MP, Henning-Boehncke W. Psoriasis. N Engl J Med, 2005;
352:1899-912.
2. Griffiths CEM, Camp RDR, Barker JNWN, Psoriasis. In: Burns DA,
Breathnach SM, Cox N, Griffiths CE, eds. Rook’s Textbook of Dermatology. 7th ed. Oxford: Blackwell; 2005; 35.1-35.69.
3. Madariaga MG, Naldi L, Chatenoud L, Khan S, Schön MP, Boehncke
WH. Psoriasis. N Engl J Med, 2005; 353:848-50.
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7. Krueger JG, Bowcock A. Psoriasis pathophysiology: Current conceptsof pathogenesis. Ann Rheum Dis, 2005; 64: 30-36.
8. Nickoloff BJ, Nestle FO. Recent insights into the immunopathogenesis of psoriasis provide new therapeutic opportunities. J Clin Invest,
2004; 113:1664-75.
9. Grone A. Keratinocytes and cytokines. Vet Immunol Immunopathol,
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10. Scarpa R, Altomare G, Marchesoni A, Balato N, Matucci Cerinic M,
Lotti T, Olivieri I, Vena GA, Salvarani C, Valesini G, Giannetti A. Psoriatic
disease: concepts and implications. J Eur Acad Dermatol Venereol,
2010; 24(6): 627-30.
11. Mehlis SL, Gordon KB. The immunology of psoriasis and biologic
immunotherapy. J Am Acad Dermatol, 2003; 49: 44-50.
12. Mendonca CO, Burden AD. Current concepts in psoriasis and its
treatment. Pharmacol Ther, 2003; 99:133-47.
Krueger JG. The immunologic basis for the treatment of psoriasis
with new biologic agents. J Am Acad Dermatol, 2002; 46: 1-23.

5

SEMNIFICATIA CLINICA A MODIFICARILOR INTERLEUKINEI-8
CIRCULANTE LA PACIENTII CU PSORIASIS ACUT NETRATATREZUMAT

Psoriazisul sub diverse forme este una dintre cele mai frecvente boli inflamatorii cronice ale pielii întâlnită pretutindeni. Interleukina-8(IL-8) este o citokină proinflamatorie. IL-8 este un chemoatractant puternic pentru neutrofile, celulele T şi bazofile.
Scopul acestui studiu a fost să comparăm producerea de IL-8 la pacienţii cu psoriazis acut cu cea de la subiecţii aparent sănătoşi şi
să investigăm relaţia dintre concentraţia IL-8 şi severitatea bolii. Concentraţia de IL-8 în ser a fost investigată la 39 pacienţi netrataţi
(28 femei şi 11 bărbaţi cu vârsta medie 33,76±4,2 ani, cu limite între 25-50 ani) cu psoriasis acut şi 12 subiecţi apparent sănătoşi (8femei şi 4 bărbaţi cu vârsta medie 34,9±7,7 ani, cu limite între 27-54 ani). Concentraţia de IL-8 a fost măsurată folosind tehnica ELISA.
Concentraţiile citokinei (pg/ml) au fost exprimate ca media probelor lucrate în duplicat. Rezultatele sunt exprimate ca media±DS.
Concentraţia de IL-8 la lotul de pacienţi a fost 934±312 pg/ml (cu limite între 629-1246 pg/ml), la lotul martor a fost 417±126 pg/ml(cu limite între 296-539 pg/ml). Rezultatele au fost analizate folosind testul Student pe perechi. Testul Wilcoxon a fost folosit pentru
analiza statistică a diferenţelor între grupuri. Valoarea p<0,05 a fost considerată semnificativă. Există creştere semnificativă de IL-8
în psoriazisul acut (p<0,01). Nu s-a găsit corelaţie între concentraţiile de IL-8 în ser şi severitatea bolii. Rezultatele sugerează că
IL-8 prezintă o importanţă fiziopatologică.
Cuvinte cheie: psoriazis, inflamaţie, IL-8 în ser

6

Fiziologia - Physiology  2011.21.2 (70)

HYPERALGESIA – AN OVERVIEWDANIELA MOTOC, NICOLETA CLARISA TURTOI, FRANCISC SCHNEIDER, VIRGIL VASCA,ELISABETA VASCA„Vasile Goldis” Western University, Arad, Romania

ABSTRACTThe current article is designed as an overview of the most recent data on hyperalgesia, emphasizing the following aspects: hyperalgesia – symptom and/or disease, molecular bases of hyperalgesia, types, pathways, cells, mediators and receptors involved,
opioid-induced hyperalgesia, the role played by inflammation, the involvement of hippocampus in the exaggerate persistent painful
sensation and the experience of hyperalgesia according to individual factors such as age, gender, anxiety.
Key words: hyperalgesia, nociception, sensitization, opioid, nocebo, inflammation.

INTRODUCTION
Pain is certainly the most common symptom of trauma or
diseases, which determines patients to seek medical assistance.
Although it seems paradoxical given its unpleasant nature, pain ismore and more considered a precious advantage that contributes
to the integrity of the human body.
Essential to the pain sensation are the nociceptors – nerve
cell endings/receptors – that send information to the central
nervous system regarding the place and the nature of the injury,
as well as input about the intensity of the pain response that is tofollow. By means of the nervous system, a stimulus of nociceptive
intensity will cause a series of events that will loop to the site of
the injury to facilitate the immune response which will influence
the severity and duration of pain.
Due to excessive mechanical, chemical or thermal stimulation, the nerve endings of myelinated and unmyelinated fibers
become activated. This aspect is of ultimate importance forsurvival, since acute nociception is one of the most intriguing
defense mechanisms of the human body. Like all the senses,
acute nociception sends information about the internal or external
environment, acting as an alarm system meant to provide vital
input for survival. A metaphor for the lack of pain is the caseof congenital insensitivity to pain that leads to a significantly
lower life expectancy due to injuries/trauma of which there is
no awareness.
At the other end, when nociception persists for a long time,
it ceases to be a symptom and it becomes a pathology in itself.
As long as pain sends a message about a threat/injury, it is avaluable protective tool. Still, when it ceases to serve this purpose
and it continues long after the threat stopped, it not only becomesuseless, but becomes pathological.
Chronic pain occurs due to the interaction of environmental
and neurogenic factors. What starts as local injury, ischemia or
inflammation determines chronic alterations of the environment,
impacting the function of the sensory nerve fibers. One of thesealterations that occur during chronic pain is a peripheral andcentral sensitization that leads to hyperalgesia and allodynia.
Definition
Hyperalgesia is increased pain sensitivity due to a decreasein threshold, an increase in suprathreshold response and spontaneous activity. It needs to be distinguished from allodynia, which
is a nociceptive reaction triggered by nonnociceptive stimuli.
Hyperalgesia is characterised by a leftward shift in thestimulus-response function. The electrophysiological correlate
for hyperalgesia is a sensitization of nociceptive neurons thatcan be peripheral, central or mixed.
Hyperalgesia – symptom or disease?
The function of the nociceptive system implies protective
responses to acute painful stimuli. This system adapts to theincreased vulnerability of the damaged tissue by lowering thenociceptive thresholds that usually happens in case of hyperalgesia. Thus, hyperalgesia per se is not negative, but a normal
reaction of the nociceptive system meant to prevent further
tissue damage.
Still, sometimes, the intensity, duration or location of pain
may not accurately reflect the threat, which is the case withhyperalgesia prolonged longer than the action of the stimulus/
damage. Also, hyperalgesia may reflect an alteration of the normal function of the peripheral or central nervous system. Thus,
when hyperalgesia ceases to be protective and starts being
maladaptive and the pain ends serving a purpose, the pain itself
becomes a disease that needs to be addressed (16).
Molecular basis
In a matter of minutes or hours, at the site of the injury or
inflammation a number of inflammatory mediators and productsthat result from tissue breakdown are released: proteases, histamine, serotonin, bradykinin, nitric oxide, prostanoids, cytokines,
ATP. This “inflammatory soup” irritates or sensitizes the nerve
endings that determines nociceptive signaling and neurogenic

Received March 24th, 2011. Accepted May 28th, 2011. Address for correspondence: Daniela Motoc, MD, PhD, „Vasile Goldis” Western University, Arad, Romania,
Feleacului Street No.1, Arad, e-mail: motocdana2005@yahoo.com

2011.21.2 (70)  Fiziologia - Physiology

7inflammation – which leads to the release of neuropeptides (14).
Through the sympathetic efferents, catecholamines are released.
This mixed inflammation, immunogenic and neurogenic, resultsin chronic pain (12).
Bradykinin induces pain and hyperalgesia that can beblocked by inhibitors of prostaglandin synthesis or by sympathectomy.
Prostaglandins play an important role in determining hyperalgesia. Still, PGE1, which plays the leading role in inducing
hyperalgesia is not found in inflammatory exudates, while PGE2,
which is found in exudates, has a significantly lower hyperalgic
effect (11).
Recent studies suggest that in inducing hyperalgesia there
are other cells that become involved, such as:•
Polymorphonuclear leukocytes (PMNLs).
Leukotriene B4 is also involved in hyperalgesia, dependent
to PMNLs. The incubation of LTB4 with PMNLs in vitro
releases a hyperalgesic factor in the supernatant which isa product of the 15-lipooxygenase pathway of arachidonic
acid metabolism (9, 10, 13).
•The sympathetic postganglionic neurons. A
number of studies suggest that sympathetic post-ganglionic neuronal activity exacerbates hyperalgesic states insome patients with nerve injury, that suffer from causalgia
or reflex sympathetic dystrophy (11).
The mechanisms and pathways that are involved in mediation of nociception are transcriptional regulated. A major role in
the regulation of the inflammatory milieu is played by the nuclear
factor (NF)-κB through the control of gene expression/suppression. An association has been established between (NF)-κB andpain/nociception, probably by regulation of the inflammatory loop
and the biosynthesis of pro-inflammatory mediators. There is anindication that the effective inhibition of this factor, the associatedupstream kinases and the pathways that regulate its nuclear
translocation could be areas of interest for a new strategy meant
to alleviate inflammation and inflammatory-related pain (6).
Types of hyperalgesia
There are three types of hyperalgesia:
1. Primary hyperalgesia is the hyperalgesia that occurs at
the site of the injury due to sensitization of nociceptive nerve
endings. Nowadays, there are ongoing studies that suggest thatan equally important part is played by an altered processing in
the central nervous system.
2. Secondary hyperalgesia occurs in an adjacent or remotearea of the injury site. It is due only to the changes in the processing of sensory information in the CNS.
3. Reffered hyperalgesia can occur in the skin, remote fromthe site of injury.
PRIMARY HYPERALGESIA. There are a number of stimuli
that can determine this type of hyperalgesia, such as heat, mechanical stimuli and inflammation.
Hyperalgesia to heat occurs after a skin lesion and after
8

inflammation. Sensitization of primary afferent nociceptors toheat account for this type of hyperalgesia. An interesting fact isthat a burn injury to the glabrous skin of the hand determines
hyperalgesia to heat, but does not lead to sensitization of
CMHs, while a burn injury to the hairy skin determines such asensitization. This suggests that, according to the skin type, theresponse may differ. Hyperalgesia to heat can develop after
inflammation. When inflammatory mediators are injected into a
receptive field of nociceptive afferents, a marked sensitization
to heat stimuli occurs.
Hyperalgesia to mechanical stimuli can also occur
both after skin injuries and after inflammation by a number of
mechanisms:
Nociceptor sensitization. After a burn injury, nociceptors
are sensitized to heat stimuli, but the mechanical threshold of
nociceptors is not lowered. Still, nciceptors are sensitized tomechanical stimuli in inflammation.
Spatial summation. If heat is applied at an edge of thereceptive field of a nociceptor, the receptive field expands into
the area of injury. Thus, a mechanical stimulus in the area of
injury evokes a response in a greater number of nociceptive
afferents, which will lead to spatial summation in the CNS thatwill determine an increased pain.
Central sensitization plays a major role in secondary hyperalgesia, but it seems to be involved to a certain level in primary
hyperalgesia to mechanical stimuli.
Inflammatory mediators
The thermal hyperalgesia associated with induced inflammation or to specific inflammatory mediators (bradykinin, ATP,prostaglandin E2, proteases) depends strongly on the presenceof TRPV1 ion channel. The activity of TRPV1 can be sensitizedby all of these mediators via a number of mechanisms.
The most direct mechanism by which mediators sensitize TRPV1 is by generating co-agonists such as protons orarachidonic acid metabolites. Other mechanisms involve direct
phosphorylation by serine/threonine kinases and the alterationof the phospholipids content of the membrane. Regardlessthe mechanism, one thing is certain: the nociceptors havedeveloped many ways to respond to inflammation by becoming
hypersensitive (2).
SECONDARY HYPERALGESIA occurs as a response tomechanical stimuli, but not to heat stimuli. There are two typesof mechanical hyperalgesia: punctate (static) and stroking(dynamic).
Punctate hyperalgesia is hyperalgesia in response to
sharp stimuli that normally generate pain (pin prick). Stroking
hyperalgesia refers to pain to a light moving touch, but not bluntpressure.
An explanation for secondary hyperalgesia is that thesensitization that starts at the site of the injury then spreads tohealthy tissue. Still, there is data suggesting that this spreading
does not necessarily take place. For instance, burn injury to a halfof the receptive field is not accompanied by sensitization in the
Fiziologia - Physiology  2011.21.2 (70)uninjured receptive field. Another interesting thing is that the areaof flare is generally smaller than the area of hyperalgesia.
While both punctate and stroking hyperalgesia present central sensitization, there are some differences between them:
1.
Punctate hyperalgesia occurs under theaction of a punctate stimuli and, compared to the stroking
hyperalgesia, the area involved is larger and the pain lasts
longer.
2.
Stroking hyperalgesia (allodynia) is triggeredby a light stroking, lasts a shorter period of time and it
involves a smaller area. (2)Opioid-induced hyperalgesia
Opiates have been used for a long time in the treatment ofchronic pain, such as the pain associated with cancer. Paradoxically, studies have shown that opiates used to treat pain can
induce hyperalgesia.
Opioid-induced hyperalgesia is a condition that is translated
clinically as hyperesthesia and allodynia. It occurs in some of thepatients that receive chronic opioid therapy. Clinical reports thatdate back to the late 19th century documented that hyperalgesia
goes hand in hand with opioid addiction. Later studies suggested
that pain sensitivity is different in subjects with opioid addictions
compared to those who are not addicted (1). Research also
indicates that hyperalgesia occurs in the context of short-termand continuous therapy in which physical dependence andwithdrawal did not play a role.
There are a number of mechanisms associated with thistype of hyperalgesia. A possible mechanism concerns the NMDAreceptor. Changes mediated via this receptor in the dorsal hornoccur after a long exposure to opioid drugs resulting in hyperalgesia. Glutamate-associated activation of NMDA-receptors
generates spinal neuron sensitization. NMDA-receptor-cellularmechanisms mediate irreversible neurotoxic changes, including
apoptosis.
Other studies have shown that hyperalgesia is closely linked
to the increased excitatory peptide neurotransmitters such as
CCK which activate spinal pathways that up-regulate spinal
dynorphin. These substances have a pro-nociceptive action,
causing central sensitization – hypersensitivity of the spinal cord
to nociceptive inputs from the periphery (4).
Tolerance and opioid-induced hyperalgesia may be indistinguishable clinically. Pharmacological tolerance, opioid
induced pain and the hyperalgesia of neuropath pain areclosely linked. It is not clear which of the phenomena produces
the clinical syndrome of opioid tolerance. Studies suggest that
this abnormal sensitivity is closely related to the mechanism bywhich neuropath pain occurs. The pro-nociceptive actions oflong-term opiate administration need a certain interaction with
opiate receptors and probably are not due to the accumulationof excitatory metabolic products. The exact mechanism by which
this event takes place remains unknown, but some recent studies
suggest that plasticity initiated by opiate receptor interaction is
involved (5). Other studies also emphasize the role played by
the pain vulnerability that occurs as a result of events that took
2011.21.2 (70)  Fiziologia - Physiology

place earlier in life (15).
Contribution of individual factors
1. Gender. In many studies it has been suggested thatwomen are more sensitive to pain than men are due to enhanced
central pain processing and psychological factors. Still, a study
performed by M.T. Jensen and K.L. Petersen shows that there
are only small differences between men and women regarding
nociception and neuronal sensitization (7).
2. Age. Because of ethical concerns, studies of pain on
children are scarce. Still, the existing data suggests that, due tothe immaturity of sensory processing in the spinal cord, children
have lower thresholds for sensitization and excitation. In the
neonate, hyperalgesia is explained partially by the substantial
upregulation of neurotrophins as a response to the damaged
tissue. This phenomenon is a few times amplified in neonatescompared to adults. Also, because of the higher plasticity in the
peripheral and central connections in neonates, early damage
during infancy can lead to structural and functional alterations in
pain pathways that persist in adulthood. Another age-related category that is at risk for hyperalgesia is the elderly. Since chronic
pain is found more often in the elderly population – two times
compared to pain in younger individuals – and its management
is often poor, it is no surprise that hyperalgesia is found more
frequent in the elderly, compared with younger adults.
3. Emotions. J. Kong et colab. performed a study on theneural mechanisms of hyperalgesic nocebo effect (also called“negative placebo effect”), since it is sometimes incriminatedas a factor for a number of symptoms and adverse reactions inmedical care. They showed, by means of functional MRI, that
there is a link between the left frontal operculum and hippocampus and the pain network – insula, operculum, ACC and left S1/M1 (8). Thus, nocebo hyperalgesia may take place through anaffective-cognitive pain pathway, with the hippocampus playing
a major part.
4. Anxiety. It has been demonstrated that the negative expectations of a subject of pain increase when an inert
substance is administered will facilitate pain transmission andinduce hyperalgesia by means of anticipatory anxiety. Throughan experimental study on ischemic pain induced to healthy
volunteers, F. Benedetti et colab showed that verbally induced
nocebo hyperalgesia is associated to a hyperactivity in the
hypothalamic-pituitary-adrenal axis. Their findings emphasisethat there is a close link between anxiety and the nocebo hyperalgesia, in which colecystokinergic systems play a major part(3). Studies have demonstrated that the analgesic placebo ismediated by endogenous opioids, while the hyperalgesic nocebo
effect is due to the opposite activation of endogenous opioidergicand CCKergic systems.
CONCLUSION
Hyperalgesia, as a symptom, is part of the intricate defense
mechanism of the human body. Still, a prolonged intense painful sensation that serves no purpose in the alarm system of the
body becomes an entity that needs to be addressed and taken
9seriously. From the three types of hyperalgesia, two are more
important: primary hyperalgesia that occurs at the site of the
injury due to the sensitization of nerve endings and secondary
hyperalgesia that takes place due to alterations in the processing of sensory information in the central nervous system. In
hyperalgesia, at a molecular basis, a major role is played by
the inflammatory mediators that constitute the “inflammatorysoup” that sensitize the nerve endings. Recently, it has been
demonstrated that polymorphonuclear leukocytes are involvedin the hyperalgesia through leukotriene B4, as well as the sympathetic postganglionic neurons. Opiod-induced hyperalgesia isa type of hyperalgesia that occur after administration of opioid
medication or drugs. Changes mediated via the NMDA-receptor
in the dorsal horn occur after a long exposure to opioid drugs
resulting in hyperalgesia.
There are a number of individual factors that alter the experience of pain in certain groups of individuals: children (neonates
included) and elderly are populations at risk for hyperalgesia.
There is little difference between hyperalgesia in men and in
women, and the few differences present are due to socio-psychological factors and to enhanced central pain processing.
The emotional life of the individual alters one’s experienceof pain. It has been shown that the left frontal operculum and the
hipppocampus are linked to the pain pathways. Also, by meansof anticipation and nocebo effect, subjects that have a higher
level of anxiety will experience pain more dramatically.
REFERENCES

1. Angst MA, Clark JD. Opioid induced hyperalgesia. Anesth., 2006;
104: 570-87.
2. Basbaum AI, Bushnell C. Science of Pain, Academic Press, Elsevier
Inc., 2009.
3. Benedetti F, Amanzio M, Vighetti S, Asteggiano G. The Biochemical and Neuroendocrine Bases of the Hyperalgesic Nocebo Effect. J
Neurosci., 2006; 26: 12014-22.

4. DuPen A, Shen D, Ersek M. Mechanisms of Opioid-Induced Toleranceand Hyperalgesia: Opioid-Induced Hyperalgesia. Pain Manag Nurs.,
2007; 8(3): 113-121.
5. Gardell LR, King T, Ossipov MH, Rice KC, Lai J, Vanderah TW,
Porreca F. Opioid receptor-mediated hyperalgesia and antinociceptive
tolerance induced by sustained opiate delivery. Neurosci Lett., 2006;
20; 396(1): 44-9.
6. Haddad JJ. Molecular Regulation of Inflammatory Pain and Hyperalgesia – Is NF-κB the Lynchpin? EXCLI Journal, 2007; 6.
7. Jensen MT, Petersen KL. Gender Differences in Pain and Secondary
Hyperalgesia after Heat/Capsaicin Sensitization in Healthy Volunteers.
The Journal of Pain, 2006; 7: 211-7.
8. Kong J, Gollub RL, Polich G, Kirsch I, LaViolette P, Vangel M, Rosen
Bruce, Kaptchuk TJ. A Functional Magnetic Resonance Imaging Studyon the Neural Mechanisms of Hyperalgesic Nocebo Effect. The Journalof Neuroscience, 2008; 28: 13354-62.
9. Levine JD, Lam D, Taiwo YO, Dontoni P, Goetzl EJ. Hyperalgesic
properties of 15-lipooxygenase products of arachidonic acid. Proc. Natl.
Acad. Sci., 1986; 83: 5331-4.
10. Levine JD, Lau W, Kwiat G, Goetzl EJ. Leukotriene B4 produces
hyperalgesia that is dependent on polymorphonuclear leukocytes.
Science, 1984; 225: 743-5.
11. Levine JD, Taiwo YO. Hyperalgesic Pain: A review. Anesth Prog,
1990; 37(2-3): 133-135.
12. Levine JD, Taiwo YO, Collins SD, Tam JK. Noradrenaline hyperalgesia is mediated through interaction with sympathetic postganglionic
neurone terminals. Nature, 1986; 323: 158-60.
13. Levine JD, Gooding J, Donatoni P, Borden L, Goetzl EJ. The roleof polymorphonuclear leukocyte in hyperalgesia. Journal of Neuroscience, 1985; 5: 3025-9.
14. Pappagallo D. The Neurological Basis of Pain, The McGraw-Hill
Companies, Inc., 2005.
15. Rivat C, Laboureyras E, Laulin JP, Le Roy C, Richebe P, Simmonet
G. Non-nociceptive environmental stress induces hyperalgesia, notanalgesia, in pain and opioid-experienced rats. Neuropsychopharmacology, 2007; 32(10): 2217-28.
16. Sandkujler J. Models and Mechanisms of Hyperalgesia and Allodynia. Physiol Rev., 2009; 89: 707-758.

HIPERALGEZIA – PRIVIRE DE ANSAMBLU
REZUMAT
Articolul de fata este menit a fi o trecere in revista a ultimelor date din literatura referitoare la hiperalgezie, subliniind urmatoarele
aspecte: hiperalgezia – simptom si/sau boala, bazele ei moleculare, tipuri, mecanisme, celule, mediatori si receptori implicati, hiperalgezia indusa de opioide, rolul jucat de inflamatie, implicarea hipocampului in senzatia dureroasa exagerata persistenta si experienta
particulara a hiperalgeziei in functie de factori individuali precum varsta, sex, nivel de anxietate.
Ca simptom, hiperalgezia este parte integranta a mecanismului complex de aparare al organismului uman. Totusi, persistenta unei
senzatii dureroase intense care nu slujeste niciunui scop in sistemul de alarma al organismului devine o entitate care trebuie solutionata
si luata in serios. Dintre cele trei tipuri de hiperalgezie, doua sunt mai importante: hiperalgezia primara, care se produce la locul leziunii
datorita sensibilizarii terminatiilor nervoase si hiperalgezia secundara, care se produce datorita procesarii modificate a informatiei
senzoriale la nivelul sistemului nervos central. In hiperalgezie, la nivel molecular, un rol important il joaca mediatorii inflamatiei, carealcatuiesc „supa inflamatorie” ce sensibilizeaza exagerat terminatiile nervoase. Recent, s-a demonstrat ca leucocitele polimorfonucleare sunt implicate in hiperalgezie prin leucotriena B4. Hiperalgezia indusa de opioide este un tip particular de hiperalgezie care se
produce dupa administrarea medicatiei opioide. Dupa expunerea prelungita la acest tip de medicatie sau dupa consumul de droguri
opioide apar modificari la nivelul cornului posterior al maduvei ce implica receptorul NMDA, rezultatul fiind hiperalgezia.
Experienta hiperalgezica este resimtita diferit in functie de varsta si starea emotionala a subiectului, un rol important in amplificarea
durerii revenind anxietatii.
Cuvinte cheie: hiperalgezie, nociceptie, sensibilizare, opioid, nocebo, inflamatie.

10

Fiziologia - Physiology  2011.21.2 (70)REVIEWPARTICULARITIES OF ASSOCIATION BETWEEN DIABETESMELLITUS AND THYROID DISEASESADRIANA GHERBON, LAVINIA NOVEANU, GEORGETA MIHALAŞ
Department of Physiology, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT
Diabetes mellitus and thyroid diseases represent two endocrinopathies frequently in general population. Because insulin and thyroid
hormones are implicated in cellular metabolism, excess or deficiency of one of these determinate functional disorders of another.
Autoimmune pathology appears by almost all endocrine glands and affects them in a variable incidence. Type 1 diabetes associatedwith certain endocrine disorders, pictures the autoimmune polyglandular syndromes.
The purpose of this review is to show epidemiological, ethiopatogenic, diagnostic and therapeutic particularities of association
between diabetes mellitus and thyroid diseases.

1. EPIDEMIOLOGY
The thyroid disorders are common in the general population;
their prevalence increases with age. The screening for thyroid
disorders is indicated in certain high-risk groups as new bornand elderly people (31). The hypothyroidism is the most common thyroid disorder in adults and older women. Its origin is
usually autoimmune, presenting as atrophic primary hypothyroidism or Hashimoto thyroiditis. It may be also secondary toradioactive iodine treatment or thyroid surgery. In rare cases,
occurs secondary hypothyroidism to hypothalamic or pituitarydisease (31). By contrast, the hyperthyroidism is less common, with a ratio female/male 9/1. The Graves’s disease ismost common and usually affects young adults. The toxic
multinodular goiter usually occurs in older people (31).
The patients with diabetes mellitus (DM) have an increased
prevalence of thyroid disease compared with non-diabetic population (31). As patients with a specific organ autoimmune disease
are at risk of developing other autoimmune diseases, and thethyroid disorders are more common in women, it is not surprising that 30% of the women with type 1 diabetes have thyroid
disease. The rate of postpartum thyroiditis in diabetic patientsis three times higher than in healthy women (31).
A number of studies also indicate a high prevalence of
thyroid disorders in patients with type 2 diabetes, the hypothyroidism is the most common disorder encountered (6). The
thyroid disorders into the general population have a prevalenceof approximately 6.6%. In patients with diabetes, the thyroid
disease prevalence is between 10.8% and 13.4%, consisting of:clinical hypothyroidism (36%), sub-clinical hypothyroidism (41%),
hyperthyroidism (12%) and postpartum thyroiditis (11%) (31).
1.1. Epidemiology of type 1 diabetes associatedwith thyroid diseases
Type 1 diabetes is commonly associated with endocrine andsystemic diseases with autoimmune etiology of type: Graves-

Basedow disease, Hashimoto’s thyroiditis, Addison’s disease,celiac disease, pernicious anemia, myasthenia gravis, vitiligo,
etc. (6). For people with type 1 diabetes, ≈1-100 patients willdevelop Graves’ disease (3) and ≈ 1-20 patients are generally
affected by hypothyroidism (8). The frequency of association between type 1 diabetes with hypothyroidism and hyperthyroidism
varies from 3.2% to 4.6% and from 0.7% to 4% (22). Addison’s
disease is extremely rare in the general population, affecting ≈1-200 patients of population with type 1 diabetes (8). Celiac disease occurs in 1 from 20 patients with type 1 diabetes, pernicious
anemia in 1 from 50 patients with type 1 diabetes (8). A particularassociation of type 1 diabetes with hypo- or hyperthyroidism ischaracteristic to polyglandular autoimmune syndrome. There
are three polyglandular autoimmune syndromes.
Polyglandular autoimmune syndrome type I (PAS - I)
is an autosomal recessive transmitted disease caused by amutation in the short arm of chromosome 21, characterized by
the triad: cutaneous-mucosal candidacies, hypoparathyroidism
and Addison’s disease.
The suffering begins in childhood; coetaneous-mucosal candidacies are the first manifestation, usually followed by hypoparathyroidism and Addison’s disease (2, 20). Type 1 diabetes occurs in lessthan 4% of affected children, but increased to 12% in adults.
• Frequency: international, PAS-I is a very rare
disorder. The largest number of patients was reported
in Finland, where the prevalence was estimated at 1 to
25.000 subjects.
• Mortality/Morbidity: appear to be equivalent to individual components of the syndrome.
• Predominance of sex: the ratio F/M is 0.8 to 1.5/1;
the numbers from 1998 indicate a ratio of 2.4/1• Age: PAS - I occur in children aged 3-5 years orin early adolescence, but always appear until the third
decade of life.

Received March 15th, 2011. Accepted April 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

2011.21.2 (70)  Fiziologia - Physiology

11Polyglandular autoimmune syndrome type II (PAS - II)(24, 4) is the most common immuno-endocrinopathies. It occurs
in adulthood, especially affecting women. It is characterized by
the appearance into the same person of two or more of the following diseases: Addison disease, Graves-Basedow disease,thyroiditis of autoimmune etiology, type I diabetes mellitus,
primary hypogonadism, myasthenia gravis, and celiac disease.
Most diseases are associated with the following HLA haplotypes:
A1, B8, DR3 (DQA1 * 0501, DQB1 * 0201) and DR4 (DQA1 *0301, DQB1 * 0302). Since most syndrome components have along prodromal phase in which auto antibodies are present before
the development of the disease, the current efforts are focused inorder to determine if immunosuppressive or immunomodulatory
medications can prevent or stop the destructive processes.
• Frequency: in U.S. about 14 to 20 per 1 million people
are affected by this syndrome.
• Mortality/Morbidity: so far, the mortality and morbidity
rate of PAS - II has not been estimated from the clinical pointof view, but it is consider that they should be equal with themortality and morbidity of individual components.
• Predominance of sex: the ratio F/M of PAS - II is
3 - 4/1.
• Age: PAS - II appears in the third and fourth decadeof life.
Polyglandular autoimmune syndrome type III (PAS - III)(3) is a PAS II syndrome but without adrenocortical involvement.
It includes a group of autoimmune disorders generally characterized by severe glandular deficiency. A quarter of the patients witha gland hypofunction also present other endocrine diseases.
This syndrome is associated with the following diseases: celiac
disease, hypogonadism, myasthenia gravis, sarcoidosis, Sjogren
syndrome, rheumatoid arthritis, gastric cancer, malabsorption
due by pancreatic exocrine deficiency, and can be classified
into three subcategories:- PAS III A - autoimmune thyroiditis with DM type1- PAS III B - autoimmune thyroiditis with pernicious anemia- PAS III C - autoimmune thyroiditis with vitiligo and/or
alopecia and/or other autoimmune diseases• Frequency: exact prevalence of PAS III is unknown.
• Mortality/Morbidity: are determined by individualcomponents of the syndrome.
• Predominance of sex: PAS III is more common in
women than in men.
• Age: PAS III is typically seen in middle-aged women,
but can occur in people of any age.
DM is associated with endocrine disorders also in otherpolyglandular autoimmune syndromes (7), found with a much
lower frequency.
Type B insulin resistance syndrome is caused by thepresence of insulin antireceiver antibodies. A third of patients
have associated autoimmune suffering as systemic lupus ery12

thematosus, autoimmune thyroid disease. Although blood glucose levels are elevated secondary extreme insulin resistance,ketoacidosis is not characteristic. The patients may present
spontaneous remissions and severe hypoglycemia (secondary
insulin-like effects of insulin antireceiver antibodies, effects
demonstrated in vitro).
POEMS syndrome includes DM, primary gonad failure,sensory and motor neuropathy, and bone lesions, hyperpigmentation.
DIDMOAD syndrome is an autosomal recessive disorderthat includes diabetes insipidus, DM, optic atrophy, deafness.
DM is usually the first manifestation in children.
Trisomy 21 (Down syndrome) is associated relatively frequent with the presence of type 1 diabetes and thyroiditis. Thissuggests that the chromosomal abnormalities influence autoimmune processes or that the susceptibility to develop autoimmune
diseases can be associated with chromosomal disorders.
1.2. Epidemiology of type 2 diabetes associated withthyroid disease
In the case of association of IGT and IFG with thyroid
disorders, they usually occur as a result of excess thyroid hormones. It was found that these associations are more common
in women. In a U.S. study shows that at patients with thyroid
disease, glucose intolerance was present in 38% of cases, andincidence of clinical diabetes was ≈ 2-3% (29,30). Other authors
have obtained a prevalence of thyroid disorders in patients with
type 2 diabetes of 2.5%, the most common endocrine disorderwere subclinical hypothyroidism (4.1%) (23).
DM type 2 is frequently associated with hyperthyroidism(Graves-Basedow disease and toxic multinodular goiter). DM
type 2 is present in 11% of patients with Graves-Basedow
disease and in 5% of those with toxic multinodular goiter. The
glucose intolerance is also frequently associated with hyperthyroidism; its prevalence is much higher compared to type 2
diabetes (72.3%). In the case of the toxic multinodular goiterincidence of impaired glucose tolerance was much higher,
namely 85%, and in the case of the Graves-Basedow diseasearound 54% (21).
Also, in the case of euthyroid diffuse goiter, its prevalencein the people with DM varies between 3.4% and 17%; being
discussed the treatment with sulfonylurea involvement in its
appearance (11).
2. ETIOPATHOGENESIS
2.1. Pathogenic mechanisms involved into the association between type 1 diabetes with thyroid endocrine
disordersAutoimmune diseases are divided into “organ specific”(autoimmune disease of the pancreatic islet, brain, thyroid,parathyroid, anterior pituitary gland, adrenal cortex, skin, ovaries,
and gastrointestinal tract) and “organ unspecific” (systemic lupus
erythematosus and pernicious anemia).
Fiziologia - Physiology  2011.21.2 (70)Autoimmune response may be then developed, either
from an organ-specific antigen, or to an antigen with ubiquitous
distribution, with a wide distribution in the body.
Both at the humans and animals, there is the possibility of
associating of many organ specific diseases. For example, DM
type 1 may be associated with the chronic gastritis/pernicious
anemia, vitiligo and thyroid disorders in PAS III, which indicatesthe likelihood of common immunological factors, genetic andenvironmental issues that arise in the pathogenesis of affectedorgans.
2.2. Genetic susceptibility
The genetic factors play an important role into individual
susceptibility at autoimmune diseases, as demonstrated bynumerous epidemiological studies. In some families there isan association of several autoimmune endocrine disorders, as
between autoimmune endocrine disorders and other systemic
immunopathies, which are more common in monozygotic twins
than in dizygotic (16).
The most autoimmune diseases have a polygenictransmission, with certain genes susceptible to their occurrence.
Appearance risk of an autoimmune disease differs from one
gene to another (10). It seems likely that the location of several
susceptible genes on the same loci predisposing the person to
the concomitant development of many autoimmune diseases(12,26).
An important role in autoimmune disease susceptibility have
HLA class I and II, some having protective role and other enablingrole in appearance of autoimmune diseases (25).
In last time was incriminated the gene which encoding
cytotoxic T lymphocyte associated with molecule 4 (CTLA-4).
The polymorphism of this gene leads to decrease of the inhibitory
signal mediated by CTLA-4, causing autoimmune diseases (1,15, and 27).
Studies in Japan have shown the role of CTLA-4
polymorphism in susceptibility of children to present type 1
diabetes and autoimmune thyroid disease associated. Thus, theG allele frequency was 63.9% in patients with type 1 diabetes; atthose with GAD + it was 72.9%. In patients with Graves-Basedow
disease their frequency was around 78.6 % (19,14).
Association of this gene polymorphism with type 1 diabetes
and autoimmune thyroid disease has been confirmed by otherstudies in Caucasian patients and in other ethnic groups(19,14).
The predisposition for developing autoimmune disease islikely caused interaction susceptible gene- protective gene, asof antigen ability to target organ damage (28,13).
It is widely recognized that at the same person may appear
more organ-specific endocrinopathies. The main diseases
characterized by the combination of several immune diseases
such are called polyglandular autoimmune syndromes. There
are three polyglandular autoimmune syndromes, PAS I andPAS II such as independent entities, while PAS III is considered
a subset of PAS II. The main characteristics of these syndromes
are described in Table I.
2011.21.2 (70)  Fiziologia - Physiology

Table I. Polyglandular autoimmune syndromes - clinical and etiopathogenic
characteristics (17)PAS IIA PAS IIB (PAS(PAS II) III)Children Adult
Adult (peak 30( p e a k < 1 0 (peak 30
years)
years)
years)
Gene AIRE
and the disease
HLAcomponents
HLA - DR3, D R 3 ,influenced by
DR4-DR4
HLA-DR/DQ
genotype
PAS I
Onset age

Genetic

Clinical symptoms
Addison disease
Hypoparathyroidism
Chronic coetaneous-mucosal candidacies
Graves diseaseHashimoto thyroiditis
Pernicious anemia
DM type 1
Gonad deficiency
Vitiligo
Active chronic hepatitis
Alopecia
Malabsorption
Celiac disease
Hypopituitarism
Myasthenia gravis

++
++

++
-

-

++

-

-

+/+ (early onset)+/++ (Female)+++ (universal)
+
+
+
-

+
++
+
++
+/+
+
+/+/-

+
++
++ (late onset)
+
+/+
+
+/-

The triggers of autoimmune process
The triggers of autoimmune process are represented byexogenous environmental factors, which acting on geneticallyprone land. Their role is demonstrated by the study of populations
genetically similar but which are living in different living conditions(18).
Infectious agents - are represented by involvementof some viruses’ type: mumps virus, varicella-zoster virus,
cytomegalovirus, reovirus type 3, and measles virus for type 1
diabetes and as rubella virus, hepatitis C virus in the case of thechronic autoimmune thyroiditis (17).
The non-infectious agents - are represented by iodine,sex, estrogen, pregnancy, certain medications and stress. The
role of iodine in the development of immune thyroid disease
is controversial. Some authors report an increased incidenceof chronic Hashimoto’s thyroiditis and of Graves- Basedow
disease with increasing of iodine intake. Other authors foundno significant correlation between iodine intake and occurrenceof autoimmune thyroid diseases (17).
The mechanism by which iodine interacts with immune
system is not known. It seems that iodine would have a modulator
effect on thyroid growth factors (17). Regarding the gender ofthe subjects, at all autoimmune thyroid diseases has been a
net predominance of females, determined primarily by genetic
factors, and then by the hormone (17). Another factor involved
could be hormonal. Thus it appears that estrogen affects immune
system by effect exerted on B and T cells (5). Pregnancy can
13induce immunosuppression, followed subsequently by immune
system hyperactivity. This is demonstrated by the relatively
frequent occurrence of Graves-Basedow disease or postpartum
thyroiditis at 3-6 months after birth (17).
The drugs can impair immunity link. The main drugs involved
in altering immune process are cytokines and interferon α. The
cytokines may exacerbate a preexisting autoimmune disease. Theinterferon α determined in predisposed patients to autoimmune
thyroid disease, occurrence of anti-thyroid antibodies, but does
not induce the formation of new autoantibodies (17).
Acute stress, as chronic one determined immune
suppresses through antigen-nonspecific mechanisms, possible,
secondary of the cortisol and CRH’s effects on immune system.
Induced immune suppression may be followed by a hyperactivityof immune system (17).
The performance of autoimmune process
Autoimmune nature of autoimmune glandular syndrome
component is supported by the following factors: the presence ofchronic inflammatory infiltrate composed mostly of lymphocytes,
association of components with expression of HLA class II genes(HLA-DR/DQ), as the presence of auto antibodies that responseto specific antigens.
There are three main classes of antigens that cause specificauto antibodies (17):• molecular surface receptors (e.g. TSH receptor
involved in Graves disease and in atrophic thyroid
disorders, insulin receptors in autoimmune insulin
resistance syndrome)• intracellular enzyme (thyroid peroxidase in Hashimoto thyroiditis and 21-hydroxylase in Addison’s disease)• proteins secreted as hormones produced by affectedorgans (thyroglobulin in Hashimoto’s thyroiditis, insulin andproinsulin in type 1 diabetes)
Autoimmune thyroid diseases symptoms are caused
by: action of thyroid auto antibodies, thyroid infiltration withlymphocytes (cell aggregation in tissues causing inflammationand cell destruction), action of cytokines released by T cells
during autoimmune process, as of suppressor T lymphocytes(CD8 +) and natural killer (NK) decrease.
The thyroid autoantibodies exert its effects either directly on
thyroid cells, or on the TSH receptor, blocking or stimulating the
production of thyroid hormones. Autoimmune thyroid diseases
may occur in the same patient at different intervals during
life. The patients with Hashimoto thyroiditis may develop later
Graves disease and the most patients with Graves’ disease
may present hypothyroidism symptoms before diagnosis.
The predominant symptoms depend on existing blood auto
antibodies (auto antibodies that stimulate TSH receptor causes
symptoms of hyperthyroidism, and those who block it symptomsof hypothyroidism).
2.3. Pathogenic mechanisms involved in association
between type 2 diabetes with thyroid endocrine disorders
The hypothyroidism reduces glucose absorption from the14gastrointestinal tract. Peripheral glucose uptake is also delayed.
At the same time, glycerol release from adipose tissue is slowed,
and availability of amino acids and glycerol for gluconeogenesis
is low.
Despite easily demonstrate of the carbohydrate metabolism
abnormalities in hypothyroidism, the clinical manifestations ofthese abnormalities are rarely obvious. The patients withhypothyroidism may have a flattened glucose tolerance curve
despite the concomitant reduction in peripheral glucose
uptake. Although hypoglycemia is sometimes stated as amanifestation of hypothyroidism, is rarely an isolated sign ofthyroid hormone deficiency, and the presence of hypoglycemia
in a patient with hypothyroidism should suggest the presence ofhypopituitarism. Occurrence of hypothyroidism in a patient withinsulin dependent diabetes can lead to decrease exogenous
insulin requirements (by decreasing insulin degradation rateand decreased appetite). These patients, however, may
have ketoacidosis. Inverse, the correction of hypothyroidism
in a patient insulin dependent usually requires insulin dose
increase (9).
The hyperthyroidism is described as an increase in thyroid
hormones, whereas the thyrotoxicosis refers to events resulting
from increases in serum thyroid hormones. Graves’ disease(known as Basedow’s disease in Europe) is the most common
cause of thyrotoxicosis, other causes including toxic adenoma,
toxic nodular goiter and thyroiditis. The features of Graves’disease include: diffuse goiter, ophthalmopathy and symptomsof thyrotoxicosis, especially nervousness, sweating, weight loss
and palpitations.
The Graves-Basedow disease may be associated with type1 diabetes in polyglandular autoimmune syndrome type II, but
thyrotoxicosis itself is diabetogenic. The glucose intolerance
occurs in 50% of cases, but frank diabetes only in 2-3% ofcases. When the hyperthyroidism occurs at insulin dependentpatients, the diabetes control deteriorates and increase insulin
requirements to about half of patients. These changes arereversible with treatment of hyperthyroidism.
Several factors are involved in glucose intolerance fromthyrotoxicosis. Thus, intestinal absorption of glucose can beenhanced through accelerating gastric emptying and increase
portal venous flow. The hepatic glucose production can beincreased due to increased gluconeogenesis, glycogenolysis,
and increased expression of glucose transporter protein GLUT-2(the main transporter of glucose efflux at the plasma membraneof hepatocytes).
Increase of basal hepatic glucose production and its
suppression impossibility by insulin is the main cause of
hyperglycemia in subjects at who were given experimental thyroid
hormones. It has also been described in liver and peripheral
insulin resistance, which probably underlies exaggerated plasma
insulin response to glucose administration, observed in somenon-diabetic subjects with thyrotoxicosis. It was also revealed
existence of β-cell dysfunction in some cases of thyrotoxicosis, byincreasing concentrations of fasting and postprandial proinsulin,which may induce diabetes through a lower insulin response to
Fiziologia - Physiology  2011.21.2 (70)increased blood glucose (9).
In thyrotoxicosis, the patients may have impaired glucose
tolerance but a DM induced only through thyroid hormones
excess is unlikely. There is much more discussion regarding
involvement of triiodothyronine (T3) deficiency in etiology of type2 diabetes. Numerous studies suggest that glucose homeostasis
is the result of synergistic regulation by T3 and insulin of a genetranscription involved in glucose and lipid metabolism. The
triiodothyronine chronic deficiency should be responsible forthe metabolic alteration from diabetes, hypertriglyceridemia,
central obesity (7).
3. DIAGNOSISThe diagnosis of thyroid dysfunction in patients withdiabetes, based solely on clinical manifestations, can be difficult.
An unsatisfactory glycemic control can cause hyperthyroidism
similar symptoms, such as weight loss despite increased
appetite, and fatigue. On other hand, the severe diabetic
nephropathy can be omitted in patients with hypothyroidism
because these patients may present edemas, fatigue, and pallor
and weight gain (31).
To further complicate the diagnostic process, unbalance DM,
with or without complications, can cause alterations of thyroid
function tests, which can occur in patients without associatedthyroid disease. The typical changes are represented by thedecrease of T3, determined by the decrease of T4 conversion
in T3, decreased serum T4, due to lower fixing on protein andlower serum TSH (31).
The most sensitive test to detect the thyroid dysfunctionis the determination of TSH, which allows the diagnosis withcertainty of the hypo-and hyperthyroidism. It also allows thediagnosis of subclinical thyroid dysfunction; characterized only
through adjusting the level of TSH, with T3 or T4 in the normal
range, the patients with this type of dysfunction is usuallyasymptomatic (31).
The thyroid dysfunction can produce significant biochemical
and clinical changes. Thus, the subclinical hypothyroidism
may increase LDLc and aggravating pre-existing dyslipidemia,
increasing the risk of arteriosclerosis. On other hand, thesubclinical hyperthyroidism may increase the risk of cardiac
arrhythmias and can exacerbate a pre-existing coronary heartdisease. Because the patients with diabetes are an increased
risk for cardiovascular disease, the diagnosis and treatment ofassociated thyroid disease is important (31).
The presence on antiperoxidase thyroid antibodies (TPO)
has a role in predicting occurrence of autoimmune thyroid
disease, particularly of the hypothyroidism. Autoantibodies may
occur long before obvious clinical manifestations of autoimmune
thyroid disease and predict the appearance of clinical disease.
The patients with polyglandular autoimmune syndrome may
present autoantibodies against the same antigen. At the patientswith present AB antiTPO must make their annual screening forearly detection of thyroid dysfunction and establishment of an
appropriate treatment (17).
2011.21.2 (70)  Fiziologia - Physiology

Also, at all the patients with diabetes should be performeda screen for adrenal autoantibodies, celiac disease, steroid,
gastric parietal and thyroid even at diagnosis. Also, at thepatients who have one of the diseases listed above must
be confirmed existence of diabetes by determining the ICA,
GAD, IA2, in absence of characteristic clinical manifestations(17).
At the family members of patients with diabetes must also
determine autoantibodies, especially if the diabetic patient has
thyroid autoantibodies. Identifying of the autoantibodies presence
should be followed by hormonal assessment and the treatmentof thyroid disease (17).

Fig.1. Diagnostic workup for the association between immune-mediated (type1) diabetes mellitus (IMD) and other autoimmune diseases (A. Kukreja and N.
MacLaren - 2005) (17)

4. THERAPEUTIC PARTICULARITIESThe clinical hypothyroidism should be treated throughhormone replacement therapy. The most used replacement
therapy is with L-thyroxin. Usual dose is 1.6 µg L-thyroxin/kg.
Often, in the cases of mild thyroid failure, initial dose may belower and it is adjusted according to TSH value at every 23
months. With the normalization of TSH levels and titrating L15

thyroxin, the TSH should be monitored annually. In the case of
aggravation thyroid failure is necessary to increase the dose of
thyroxin. The L-thyroxin therapy in subjects with diabetes may
exacerbate a pre-existing coronary artery disease, by increasing myocardial contractility and heart rate. Therefore, it is betterto start with a low dose, around 25 µg/day, followed by a slow
growth, monthly with 25 µg /day, monitoring of clinical status
and TSH level (31).
The subclinical hypothyroidism should be treated inpatients who present increased LDLc, which is worsened byhypothyroidism, which present the AB antiTPO detectable inserum (which means a possible transition to clinical hypothyroidism) and in patients with clinical manifestations (31).
The hyperthyroidism can cause adverse effects suchas 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 theGraves-Basedow disease long-term remission is less than 40%.
The patients with toxic multinodular goiter or with autonomous
thyroid nodules should be treated definitive with iodine radiotherapy or surgery (31).
The key of the successful management of the 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 (17).
Educating patients regarding the nature of the disease isoften critical for early recognition of associated autoimmune disorders, and as in any chronic disease, the psychosocial supportshould be evaluated individually (17). The genetic counseling is
also justified at affected family members who required carryingout specific tests (28).
Emergency identification should be performed in all patientswith polyglandular autoimmune syndrome because use of highdoses of corticosteroids in acute stress usually prevent adrenal
crisis in the patients with Addison’s disease, in those with adrenal
auto antibodies and increased risk of adrenal insufficiency. Someauthors believe that exogenous supplement with glucocorticoids
administered during acute stress is indicated also at asymptomatic individuals with biochemical changes of asymptomaticadrenocortical disease (17). From all endocrine components ofthe polyglandular autoimmune syndrome, only diabetes has nota favorable prognosis after hormonal replacement therapy andappropriate monitoring (17).
Due to long-term vascular complications, the diabetes is acandidate for aggressive therapeutic interventions. For example,
the studies results which have used cyclosporine A and azathioprine for the newly diagnosed diabetes treatment have demonstrated some metabolic benefits, but not for long term, even with
continued immunotherapy. As a result, any immunomodulating
therapy should be considered experimental and should be prescribed only in the controlled trials. Consequence of identification
16of new auto antigens and better knowledge of the pathogenesisof the disease, selective therapies may be introduced that does
not cause generalized immunosuppression (17).
Also, in the near future is predicting curative organ transplantation. The transplantation of pancreas or pancreatic islands
has been used in patients with diabetes and chronic kidney disease. Adrenal gland transplantation was successfully performed
in experimental rodents and humans (17).
In conclusion, thyroid damage is common in the patientswith diabetes and may cause significant metabolic changes.
Therefore, thyroid function regular screening is indicated in all
the patients with diabetes for establishment of early treatmentof subclinical thyroid dysfunction (17).
The best screening test is the TSH determination. At thepatients with type 1 diabetes is useful to determine the presenceof anti-TPO antibodies. If they are present, it is indicate annual
determination of TSH. In their absence, the TSH determination
should be performed at every 2-3 years (17).
At the patients with type 2 diabetes, the TSH determinationis performed at diabetes diagnosis and then at a minimum 5
years interval (17).
In the case of the patients with thyroid disease is obligatory
annual determination of glycemia. In the case of elevated blood
glucose levels, according to the values obtained, it is necessary employment in one of the category: type 1 diabetes, type2 diabetes or IGT (17).
If the thyroid disease is on autoimmune nature it is betterto determine the diabetes specific antibodies for identify thepresence of the type 1 diabetes (17).

Fig.2. Algorithm of treatment of different forms of diabetes (by a. Kukreja andn. Maclaren - 2005) (28)

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PARTICULARITATI ALE ASOCIERII DINTRE DIABETUL ZAHARAT
SI AFECTIUNILE TIROIDIENEREZUMAT
Diabetul zaharat şi afecţiunile tiroidiene reprezintă două endocrinopatii frecvent întâlnite în populaţia generală. Deoarece insulina
şi hormonii tiroidieni sunt implicaţi în metabolismul celular, excesul sau deficitul unuia dintre aceştia determină tulburări funcţionale
ale celuilalt.
Procesele autoimune interesează aproape toate glandele endocrine, care sunt afectate într-o incidenţă variabilă. Diabetul zaharat
tip 1 asociat cu anumite endocrinopatii realizează tabloul poliendocrinopatiilor autoimune.
Scopul acestei treceri în revistă este de a arăta particularităţile epidemiologice, etiopatogenice, diagnostice şi terapeutice ale asocierii
dintre diabetul zaharat şi afecţiunile tiroidiene.

2011.21.2 (70)  Fiziologia - Physiology

17

THE EFFECTS OF HYPOXIA ON THE PROLIFERATION AND
APOPTOSIS OF HUVEC CULTURES IN VITROCRISTINA MARIA VAIDA VOEVOD, IOANA BALDEA*, ADRIANA MURESAN
Department of Physiology, University of Medicine and Pharmacy ”Iuliu Haţieganu” Cluj-Napoca

ABSTRACT
Objective: Endothelial cells that line blood vessels are anatomically positioned at the interface of the blood and tissue exchange, andthus, endothelial cells are especially influenced by hypoxemia. The impact of hypoxia on endothelial cells is complex and includes
changes in metabolism, gene expression, and induction of specific cell surface proteins. With this aim, we investigated the effects
of anoxia on the proliferation rates and apoptosis of HUVEC cell cultures.
Methods: Cell source: Commercial human umbilical vein endothelial cells (HUVEC) were bought from European Collection of Cell
Cultures (ECACC,Porton Down,Salisbury, UK) and multiplied in standard medium: RPMI, supplemented with 10% fetal calf serum(FCS), gentamicin 50µg/ml, amphotericin 100µg/ml (Biochrom AG, Germany). Cell cultures in the 23rd to 28th passages were used.
Hypoxia exposure of the HUVEC cells. Growth medium was replaced with fresh medium equilibrated with hypoxic gas mixture, andcells were placed in the hypoxic chamber () to a gas mixture of 5%CO2, 95% N2, humidified with water vapors, at 37°C for 24h,
respective 72h. Cytotoxicity assay was done using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega Corporation, Madison, USA). Apoptosis induction assessment was done using Annexin V-FITC staining (BD Pharmingen, USA).
Results: Hypoxia exposure diminished the proliferation rate, in a time dependent manner (p hypoxia 24h versus hypoxia 72h, p=
5,544744E-2). The inhibition of proliferation was significant when compared to the controls (p hypoxia 24h versus control 24h, p=
9,3E-4; respective p hypoxia 72h versus control 72h, p= 3.08209E-12). The effect of hypoxia on cell proliferation at 24h was not
cytotoxic, with a diminished proliferation rate with only 5.7%, however 72h of hypoxia exposure greatly diminished the proliferation,
having a citotoxic effect, with a 27% of proliferation rate reduction. 72h of hypoxia exposure greatly diminished the cell viability, thus
the cells lost the surface adherence and were washed away during the annexin V staining procedure. This explaines why the % of
apoptotic cells is diminished at 72h of exposure when compared to 24h of hypoxia.
Conclusions:������������������������������������������������������������������������������������������������������������������������
Hypoxia and reoxygenation of the HUVEC cultures were stressful events that significantly decreased the cells proliferation rate, viability, induced morphological changes in the exposed cultures, and increased apoptosis.
Key words: hypoxia, HUVEC cell culture, apoptosis, proliferation.

INTRODUCTION
Tissue hypoxia frequently accompanies a variety of vasculardiseases, including thrombosis, atherosclerosis and ischemia/
reperfusion injury. Endothelial cells that line blood vessels areanatomically positioned at the interface of the blood and tissue
exchange, and thus, endothelial cells are especially influenced
by hypoxemia. Because of this critical location, endothelia
serve as a buffering monolayer during episodes of decreased
oxygen tension. Thus, the ability of these cells to tolerate andrespond to hypoxia is critical to their survival. The impact ofhypoxia on endothelial cells is complex and includes changes
in metabolism, gene expression, and induction of specific cellsurface proteins (1). Moreover, hypoxia often occurs in conjunction with, or as a direct result of, other inflammatory processes.
Under such conditions, the endothelium is bathed in a myriad ofsoluble factors, including cytokines, bioactive lipids and bacterial

endotoxines (2).
Experimental and clinical studies indicate that hypoxia plays
a fundamental role in the pathogenesis of a variety of diseases,
including cardiovascular, hematological and pulmonary disorders
and cancer (3, 4, 5)
MATERIAL AND METHODS
Cell source: commercial human umbilical vein endothelial
cells (HUVEC) were bought from European Collection of Cell
Cultures (ECACC,�������������������������������������������
Porton Down, and Salisbury, UK������������
) and multiplied in standard medium: RPMI, supplemented with 10% fetal
calf serum (FCS), gentamicin 50µg/ml, amphotericin 100µg/
ml (Biochrom AG, Germany). Cell cultures in the 23rd to 28th
passages were used.
Hypoxia exposure of the HUVEC cells. Growth medium
was replaced with fresh medium equilibrated with hypoxic gas
mixture, and cells were placed in the hypoxic chamber to a

Received 15 February 2011. Accepted 20 March 2011. Address for correspondence: Zsuzsanna Máté, University of Szeged, Faculty of Medicine, Department of
Public Health, Dóm tér 10., Szeged, Hungary, Tel. +36-65-545-119, Fax. +36-62-545-120, email: matezs@puhe.szote.u-szeged.hu

18

Fiziologia - Physiology  2011.21.2 (70)

gas mixture of 5%CO2, 95% N2, humidified with water vapors,
when compared to the controls (p hypoxia 24h versus control
at 37°C for 24h, respective 72h. The gases composition was
24h, p= 9.3E-4; respective p hypoxia 72h versus control 72h, p=
measured using an gas cromatograph (Shimazu, Japan) and3.08209E-12). The effect of hypoxia on cell proliferation at 24h
the gas flow was continuously monitored using a flowmeter.
was not cytotoxic, with a diminished proliferation rate with only
Cells were then allowed to reoxygenate for 2h in standard cell
5.7%, however 72h of hypoxia exposure greatly diminished thecultureconditions.
havinga cytotoxic effect; with a 27% of proliferation
m: RPMI, supplemented with 10% fetal calf serum (FCS), proliferation,
gentamicin
50µg/ml,
Cytotoxicity assay was done using CellTiter 96® AQueousrdth rate reduction.
0µg/ml (BiochromAG, Germany). Cell cultures in the 23 to 28 passages were used.
Non-Radioactive Cell Proliferation Assay (Promega Corporaosure of the tion,
HUVEC
cells.
Growthmedium was
Madison,USA).
All the experiments
werereplaced
conductedwith
in fresh medium equilibrated
4chamber to a gas mixture of 5%CO2, 95%s mixture, and
cells
were
placedinthehypoxic
triplicate. The cells seeded at a density of 10 /well in ELISA 96
with water vapors,
at titration
37°C for24h, plaques
respective
72h. The gases
wells micro
flat bottom(TPP, Switzerland),were composition was measured
for 24h atJapan)
37ºC andand
5% CO
in
a
humidified
atmosphere,
omatograph settled(Shimazu,
the
gas
flow
was
continuously
monitored using a2
then
incubatedinhypoxicconditionsfor24,
respective
72h.
s were then allowed to reoxygenate for 2h in standard cell culture conditions.
Duplicate
cultures,maintained
standard growth
conditions
assay was done
using
CellTiter96® inAQueous
Non-Radioactive
Cell Proliferation Assay
were
used
as
controls.
HUVEC
cultureswere
thenexposedtooration, Madison, USA). All the experiments were conductedin triplicate. The cells
20µl of MTS/PMS mixture in 100µl fresh medium/well, for 2h,4
ity of 10 /well in ELISA 96 wells micro titration flat bottom plaques (TPP, Switzerland),then absorbance at 490nm was measured using an ELISA plater 24h at 37ºC
and(Tecan,
5% Austria).
CO2 in a humidified atmosphere, then incubated in hypoxic
reader.
4, respective 72h.
Duplicate
cultures,maintained
standard
Apoptosis induction
assessment
was doneinusing
Annexin growth conditions were used
UVEC cultures
then
20µlCellsof seeded
MTS/PMS
mixture in 100µl fresh
V-FITCwere
staining(BD exposed
Pharmingen,toUSA).
at a
or 2h, then absorbanceat 2 490nm
was
density of 104/cm
were settled
for measured
24h at 37ºC using
and 5% an
CO2ELISA plate reader. (Tecan,
in a humidified atmosphere, then incubated in hypoxic conditions for 24h,
respective
72h. Duplicate
cultures,maintainedinduction assessment
wasdone using
Annexin
V-FITC
staining(BD Pharmingen, USA).
4
2 growth conditions were used as controls. Cells werestandarda density of 10 /cm were settled for 24h at 37ºC and 5% CO2 in a humidified atmosphere,
then washed and stained with Annexin V-FITC, according to then hypoxic conditionsfor 24h, respective 72h. Duplicate cultures, maintained in standard
manufacturer’s instructions, then observed under fluorescentns were usedascontrols.
Cells
thenwithwashedandE330
stained with Annexin V-FITC,
microscope (Olympus
BX41wereequipped
an Olympus
Fig.2.
Apoptosis
in the HUVEC
cultures following exposure to hypoxia for 24h res
e manufacturer’s
instructions,thenobserved
underfluorescent
microscope(Olympus
camera driven by software Olympus Master version 1.41EX)induction
Fig.2. version
Apoptosis inductionin the HUVEC cultures following exposure towith an Olympus
E330cells
cameradrivenbyby
Olympus Master
1.41EX)(6,7). Apoptotic
were scoredeye,software
at a magnification
hypoxia for 24h respective 72hcells were scored
by at
eye,
a magnificationof were40× examined.
and atleast 200
per cultures
conditionof 40× andleastat200
nuclei per condition
Comparative
imagesof nuclei
HUVEC
between the phase contrast and gtaken (8,9). microscopy after exposure to hypoxia; a, b-controls,; c,d- hypoxia exposure for
Pictures werePictures
takenwere(8,9).
Fig.2. Apoptosis induction inComparative
the HUVEC
culturesfollowing
exposure
imagesof HUVEC
culturesbetween to
thehypoxia
phase for 24h resp
exposure for 72h; apoptotic cells
areandshownin fluorescentgreen;
the cells
contrast
green
fluorescencemicroscopy
after
exposure
to undergoin
RESULTShypoxia;a, b-controls,;
c,d-thehypoxia
exposure
24h;hycontrols Comparative(b)are in a limited
number,comparedtoimagesfrom
24he,fhypoxia(d),whigimagesof HUVEC
culturesbetweentheforphase
contrast
andpoxia
exposurefor
72h;apoptotic
cells
are
shown
in
fluorescentofmicroscopy
cells is comparative(a, c); after
72h of hypoxia
exposure,
total cellexposure
number isafter exposure
to hypoxia;
a, b-controls,;
c,d-thehypoxia
forde
green; the cells undergoing apoptosis in the controls (b)are in awithadiminishednumberofapoptotic
cells(f);apoptotic
bodiesareshown
byexposure for 72h; apoptotic cells
shownin fluorescentgreen;
the cells undergoin
limited arenumber,comparedto the images from
24h hypoxia(d),magnification
through
an fluorescent
Olympus
BX41
microscope.
controls (b)are20x,
in aphotos
limitedtaken
number,theis comparative
images
from
24h
whil
while
the comparedtotal number
oftocells(a, c);
after hypoxia(d),72hofof cells is comparative (a, c); hypoxia
after 72hof hypoxia
exposure,
the totalcell(e),number is dee
exposure,
the total cellnumber is deeply
decreaseddiminished number
of apoptotic
cells(f); apoptotic
with a diminished number withof aapoptotic
cells(f);apoptotic
bodiesbodies
are shown by
are shownby arrows,
original magnification
20x,BX41
photos taken
magnification 20x, photos taken
through
an fluorescent
Olympusmicroscope.
through an fluorescent Olympus BX41 microscope.

1. HUVEC proliferation
after
exposure
the cultures
to hypoxia
different periods of time
Fig.1. HUVEC
proliferation
afterofexposureof the cultures
to hypoxiafor
for different periods of time

Fig. 3. Detailed images of the HUVEC cultures exposed to hypoxia for 24h, which underwen

e 1. The cultures significantly proliferated with time elapsed (p control 24h versus control
Comparative
images of HUVEC cultures between the phase contrast and gseen in Figure
1. The cultures
significantly proliferatedE-12). Hypoxia As
exposure
diminishedthe proliferation
rate, in a time dependent manner (pmicroscopy
after
exposure
hypoxia;a-phasecontrast,
b-greenfluorescence;
ar
Fig.
3.
Detailed
imagestoofwas
the
HUVEC
culturesexposedto hypoxia
for 24h,which underwen
timep=
elapsed(p control 24h
versus72h,
p=of
3.09398Esus hypoxiawith
72h,5.544744E-2).
The control
inhibition
proliferation
significant
whenFig. 3. Detailed
images of the
HUVEC cultures exposed to hypoxia for 24h,presenceof
apoptotic
thecultureexposedtohypoxiashowedpleiomorphismw12). Hypoxia exposure diminishedthe proliferation
rate, inbodies;aunderwent apoptosis
controls (p hypoxia 24h versus control 24h, p= 9.3E-4; respective p hypoxia 72hwhich
versustime dependent manner (p cells;
hypoxia round
24hComparative
versushypoxia72h,
imagesof
HUVEC
culturesbetweenthephasecontrast
andcells thatatexhibited
retractionof withcytoplasmic
elongations
and loss of surg
Comparative
imagesof HUVEC
culturesbetween the phase
3.08209E-12).
The effect of hypoxia oncell proliferation
24h was not
cytotoxic,ap= 5.544744E-2). The inhibitionofproliferation
wassignificant
microscopy
afterbigger
exposuretohypoxia;
contrast,
fluorescence;
ar
well
as72h
flattened,
cells which
apoptotic
bodies.b-green
contrast
andgreena-phasefluorescence
after exposuretoiferation rate with only 5.7%, howeverof hypoxia
exposuregreatlyshoweddiminishedthe microscopy
presenceof
apoptotic
bodies;thecultureexposedtohypoxiashowedpleiomorphismwving a cytotoxic effect; with a 27% of proliferation rate reduction.
round cells that exhibited retraction of cytoplasmic elongations and19 loss of surf
2011.21.2 (70)  Fiziologiacells;- Physiology
well as flattened, bigger cells which showed apoptotic bodies.

Detailed images of the HUVEC cultures exposed to hypoxia for 24h, which underwent apoptosis
hypoxia; a-phase contrast, b-green fluorescence; arrows indicate
exposed to hypoxia for 12, 24 and 36 h, showed decreased prothe presence of apoptotic bodies; the culture exposed to hypoxia
liferation rates assessed through Brdu method when comparedtive images showedof HUVEC
cultures between the phase contrast andgreen fluorescencepleiomorphism with normal shaped cells; round cells
to normoxia treated cells. Significant differences in proliferationer exposure thatto exhibitedhypoxia;retraction
a-phasecontrast,
b-greenfluorescence;arrowsindicatethe 24 hours hypoxic group and theof cytoplasmic elongations and loss
rates were foundonly between
ptotic bodies;ofthe
cultureexposedtohypoxiashowedpleiomorphismwithnormalshapedsurface adherence, as well as flattened, bigger cells which
control group (11).
s that exhibited
retractionof cytoplasmic elongations and loss of surface
adherence,
showedapoptotic bodies.
Exposure
to hypoxia as
for 24 h, respective for 72 h of the
HUVEC’s allowed viability of 90% of the culture compared to, bigger cells which showed apoptotic bodies.
the initial number of cells. This shows that the culture has theability to recover from stress, after relatively long periods ofhypoxia exposure.
The HUVEC cultures showed a peak of apoptosis induction
after 24h of hypoxia induction (figure 2 c,d), with a decreased
Annexin V-FITC staining in the cultures exposed to hypoxia for
72h (figure2 e,f). Annexin V staining reveals the early apoptosis,
thus if the cells are already experiencing the late stages of theactivation of cell death, the staining will show false negative
results. Moreover, this can be partially explained by increased
number of cells which lost their adherence to the plaque surface
and were discarded
withcellthe medium after 72h of hypoxia expocells after exposure to hypoxia, apoptosis was assessed as a % of apoptotic cells comparedto the total
Fig. 4. Apoptotic cells after exposure to hypoxia, apoptosos was assessed as
sure(figure
2.
e).
This
is
showed by the diminished number of
number to the total cell number
a % of apoptotoc cells comparedcells in the contrast phase image (figure 2. e).
HUVEC cultured in 1% oxygen followed by reoxygenation
72h of hypoxia exposure greatly diminished the cell viability,
activated the classical complement pathway resulting in C3 depothus the cells lost the surface adherence and were washed away
sition. There was an increase in apoptotic cells in these culturesduring the annexin V staining procedure. This explaines whythat was demonstrated by binding of fluorescein isothiocyanatethe % of apoptotic cells is diminished at 72h of exposure when
Annexin V and staining for hypodiploid nuclei (12).
compared to 24h of hypoxia.
This is consistent with the data reported by others. HUVEC plated onto fibrin gels in low-serum culture medium underwent rapid and
DISCUSSIONS
profound morphological changes within 12 to 48 hours of hypoxia;
Our cultures were exposed to anoxia conditions for 24h, retheir characteristic cobblestone organisation was transformed into
spective 72 h, and then allowed to re oxygenate for 2 h in normal
a network of cord-like or tube-like structures. It is likely that hypoxia
cell growth conditions. These experimental conditions are differinduces modification in the factors that integrate matrix information
ent compared to the studies in literature (10, 11, 12, 13), sinceand cytoskeletal organisation in order to contract fibrin (13).
other authors used a very low amount of oxygen atmospheresurrounding the cells. By these conditions we tried to create a
CONCLUSIONS
very stressful environment for the cell cultures, comparable toHypoxia and reoxygenation of the HUVEC cultures werethat encountered in areas that suffer a dramatic decrease ofstressful events that significantly decreased the cells proliferation
blood supply, such as infarctation or thrombosis. Moreover, werate, viability, induced morphological changes in the exposed
were interested in finding the effects of re oxygenation on thecultures, and increased apoptosis.
survival and proliferation capabilities of our cultures since they
Even if the viability of the cells was altered, there remained
are directly involved in recuperation of these events.
a relatively high amount of viable cells that could be able to supAs expected, hypoxia had an inhibitory, time dependent
port proliferation of the culture, thus recovering, when normal
effect on HUVEC cells proliferation. This effect was due partly toconditions are reestablished.
the re oxygenation, because we allowed the cells to recover from
REFERENCES
hypoxia for 2h in the humidified incubator at 37C, after which,
1. Stevens T, Rodman DM. Endothelium, 1995; 3: 1-11.
the MTS test was performed. As expected the cell proliferation
2. Pober JS, Cotran RS. Transplantation, 1990; 50: 537-541.
in the controls was maintained in cell growth conditions. This is
3. Carmeliet P. Angiogenesis in health and disease. Nat. Med., 2003;
a positive indicator for the cultures ability to multiply in normal
9: 653-60.
conditions and underlines the importance of lack of oxygen in
4. Pouysségur J, Dayan F, Mazure NM. Hypoxia signalling in cancer
the cell proliferation inhibition.
and approaches to enforce tumour regression. Nature, 2006; 441:
437-43.
This is supported by other studies which showed that HU5. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role
VEC’s exposed to hypoxia experienced decreased proliferation,of the HIF system. Nat. Med., 2003; 9: 677-84.
reduced S-phase ratio, and increases in apoptosis under the
6. Bossy-Wetzel E, Green D. Detection of apoptosis by Annexin V
condition of hypoxia (10).
labeling. Methods. Enzymol., 200; 322: 15-18.
In a study done by Bednarek et al., early passage HUVEC’s
7. Van Engeland L, Nieland JW, Ramaekers CS, Schutte B, Reutel-

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ingsperger CPM. Annexin V-affinity assay: A review on an apoptosis
detection system based on phosphatidylserine exposure. Cytometry,
1998; 31: 1-9.
8. He Z, Ma WY, Hashimoto T, Bode AM, Yang CS, Dong Z. Inductionof apoptosis by caffeine is mediated by the p53, Bax, and caspase 3
pathways. Cancer Res, 2003; 63: 4396-401.
9. Santosh KK, Anshu MR, Manjeshwar SB. Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax,
cytochrome c release, and caspase activation. Mol Cancer Ther.,
2005;4: 207-216.
10. Pengfei Zhu, Yanxia Ning, Liangqing Yao, Mo Chen, Congjian
X. The proliferation, apoptosis, invasion of endothelial-like epithelial

ovarian cancer cells induced by hypoxia. J Exp Clin Cancer Res.,
2010, 29: 124.
11. Bednarek W, Czekierdowski A, Kraczkowski J, Kotarski J. The
influence of hypoxia on the proliferation of endothelial cells originating from human umbilical vein (HUVEC)--an in vitro study. Ginekol
Pol., 2001; 72:1567-71.
12. Mold C, Morris CA. Complement activation by apoptotic endothelial cells following hypoxia/reoxygenation. Immunology., 2001;
102:359-64.
13. Baffert F, Usson Y, Tranqui L. Effects of prolonged exposure tohypoxia on morphological changes of endothelial cells plated on fibrin
gel. Eur J Cell Biol., 2001; 80:78-86.

EFECTELE HIPOXIEI ASUPRA PROLIFERARII SI APOPTOZEICULTURILOR HUVEC IN VITROREZUMAT
Obiectiv: Celulele endoteliale sunt pozitionate la interfata dintre sange si tesuturi, astfel, fiind influentate in mod special de hipoxemie.
Impactul hipoxiei asupra celulelor endoteliale e complex si include modificari in metabolismul celulelor, expresia genica si a proteinelorde suprafata. Cu acest scop am investigat efectele anoxiei asupra proliferarii si apoptozei culturilor de celule HUVEC.
Metoda: am utilizat celule endoteliale umane-linie comerciala (HUVEC – human ombilical endothelial cells) aflate in pasaje 22-28.
Anoxia a fost realizata prin incubarea celulelor cu un amestec gazos care contine 5%CO2, 95% N2, in atmosfera umeda, la 37°C.
Evaluarea proliferării celulelor s-a făcut cu Cell Titer Aqueous 96R (Promega, Madison, SUA). Evaluarea apoptozei celulare s-a facutprin coloratie cu Annexina V (BD Pharmingen).
Rezultate:expunerea la hipoxie diminueaza rata de proliferare, intr-o maniera dependenta de timp (p hipoxie 24h versus hipoxie
72h, p= 5,544744E-2). Inhibitia proliferarii a fost semnificativa statistic la celulele mentinute in mediu anoxic comparativ cu celulele
expuse la normoxie (p hipoxie 24h versus control 24h, p= 9,3E-4; respectiv p hipoxie 72h versus control 72h, p= 3.08209E-12). Efectul
hipoxiei asupra ratei de proliferare la 24h nu a fost citotoxic, cu o scadere a ratei de proliferare de doar 5,7%, totusi expunerea timp
de 72h la hipoxie a avut efect citotoxic, cu o scadere a ratei de proliferare de 27%.
Expunerea timp de 72h la anoxie a redus dramatic viabilitatea celulara, astfel incat celulele si-au pierdut aderenta si au fost spalate
in cursul procedurii de fixare a anexinei. Aceasta explica scaderea procentului de celule apoptotice la 72h comparative cu 24h.
Concluzii: hipoxia si reoxigenarea culturilor de celule HUVEC sunt evenimente stresante care reduc semnificativ rata de proliferare
celulara, viabilitatea, induc schimbari morfologice si amplifica apoptoza.
Cuvinte cheie: hipoxie, culturi de celule HUVEC, apoptoza, proliferare.

2011.21.2 (70)  Fiziologia - Physiology

21

EXPRESSION AND SIGNIFICANCE OF KI-67 ANTIGEN IN
PULMONARY CANCER. RELATIONSHIP BETWEEN KI-67 AND
OTHER PROGNOSTIC FACTORSMIRELA GRIGORAŞ1, ELENA LAZĂR1, ALIS DEMA1, MARIOARA CORNIANU1,ALEXANDRA FAUR1, LAURA S. GOŢIA2
1
Department of Pathology, University of Medicine and Pharmacy “Victor Babeş” Timişoara
2
Department of Physiology, University of Medicine and Pharmacy “Victor Babeş” Timişoara

ABSTRACT
We assessed the proliferative activity in 62 primary pulmonary carcinomas by: determining the percent of Ki-67 immunoreactive cells(stained with MIB-1 antibody), comparing Ki-67 score with the fraction of PCNA positive cells (marked with PC10 antibody) and by
evaluating nucleolar organizer regions (NORs) using the histochemical method of staining with coloidal silver and comparing Ki-67
score and the fraction of PCNA positive cells with the mean number of AgNOR per nucleus.
We followed the relationship between Ki-67 growth rate, p53 immunoreactivity and the level of epidermal growth factor receptor(EGFR), by staining sections with anti-p53 (DO-7) and anti-EGFR (31G7); tissue sections fixed in formalin and embedded in paraffinwere immunohistochemically stained using the Avidin Biotin Complex (ABC) method.
The results obtained show: (1) a Ki-67 positivity rate significantly correlated with histological grade (G3) of the tumor (p<0.001; ES) andhigh cellularity (C3); (2) a statistically insignificant relationship between Ki-67 expression and lymph node involvement (p=0.924448;
NS), invasion of pleura and/or thoracic wall (p=0.825267; NS), local relapses (p=0.581481; NS) and distant metastases (p=0.646270;
NS); (3) prevalence of p53 positive expression in poorly differentiated pulmonary tumors, with high proliferation rate (p<0.001; ES); (4)an increased level of EGFR expression in highly proliferative tumors, diagnosed in an advanced stage of the disease, which reflects
the invasive character and high potential for metastases of Ki-67+ EGFR+ pulmonary carcinomas; (5) a weak correlation between
mean of AgNOR per nucleus and IHC markers of proliferative activity.
The Ki-67 and PCNA positivity rate, correlated with cellularity of the tumor, together with clinical stage and histological differentiation,offer useful information in anticipating the evolution and prognosis of pulmonary cancer.
Key words: lung carcinoma, Ki-67, PCNA, immunohistochemistry.

INTRODUCTION
Current methods for estimating tumor malignancy andprognosis of patients with pulmonary cancer also refer to theproliferation rate of tumor cells. Proliferative activity of a tumor
can be investigated through various methods, among which
the immunohistochemical (IHC) ones proved to be a simple
and credible tool in assessing cell proliferation (Veronese SM,
1993; Yoshida YA, 1994).
Ki-67 antigen is a marker of cell cycle and proliferation, commonly used to estimate proliferation coefficient of a cell population (Seigneurin DG, 1991). It indicates growth fraction and thenumber of cells that can be found in the active division (Yoshida
YA, 1994). Ki-67 is a protein doublet with molecular weight of
345 and 395kDa (Seigneurin DG, 1991; Rowlands D, 1994),
respectively, being part of the chromosome coating layer.
In most malignant tumors, the percentage of Ki-67 positive
cells can be correlated with aggressiveness or tumor progression
parameters; this gives its practical interest in tumor histopathology, although its biochemical nature and function are still little
known (Seigneurin DG, 1991).
Ki-67 is a nuclear antigen expressed in all phases of cell
cycle, except G0 (Jasani B, 1993); it is detected during phases
G1, S, G2 and M of the cell cycle and is not evident in transit

cells from pausing state to G0 (Seigneurin DG, 1991; Martin
B, 2004).
Because in pulmonary cancer data referring to the prognostic value of Ki-67 proliferation index are little and the relationshipbetween Ki-67 positive rate and prognosis is still unclear, thispaper wants to analyze by means of IHC the proliferative activity
on a group of 62 pulmonary carcinomas; we present aspects ofcorrelating the Ki-67 score with histological subtype of the tumor,
cellularity and differentiation degree, as well as with other IHC
markers that assess proliferation activity.
MATERIAL AND METHODS
The studied material represented by pulmonary biopsies
obtained by bronchoscopy and surgical resection pieces, wasmade up of 62 cases from patients with lesions that were clinicallydiagnosed as pulmonary displasias or cancer. The entire histological material was fixed in 10% formalin and included in paraffin.
The common morphologic investigation was made on sections stained with hematoxylin-eosin and tricrom Gomori, which
helped in classifying and establishing the differentiation degreeof the lesions.
We assessed the proliferative potential of the studied
lesions by identifying the nuclear antigen Ki-67, applying theReceived Februaru 10th, 2011. Accepted May 20th, 2011. Address for correspondence: Mirela Grigoras, MD, Department of Pathology, “Victor Babes” University of
Medicine and Pharmacy Timisoara, Romania, Eftimie Murgu Square No. 2A, 300041, Timisoara, phone/fax: +40256220479; e-mail: scr-stin@umft.ro22

Fiziologia - Physiology  2011.21.2 (70)

IHC staining method of the Avidin-Biotin Complex (ABC). After
pretreatment by boiling for 60 minutes at 80-90°C in citrate buffer pH6, sections were incubated over night at 4°C with MIB-1
primary antibody (MIB-1 – Dianova, Hamburg, Germany) diluted
1/800. Visualization with AEC (3-amino, 9-etilcarbazole), followed
by a light counterstain of the nuclei with hematoxylin, allowed
highlighting of Ki-67 in the form of a final reaction product, red
and with nuclear localization.
In what concerns the proliferative activity, on the same
number of cases we tested a variety of antibodies with specificity for p53 protein (DO-7, DAKO, Glostrup, Denmark – diluted
1/500) and for proliferation cell nuclear antigen (PCNA) (PC10 –DAKO, Glostrup, Denmark – diluted 1/800) and we identified thenucleolar organizer regions (NORs) applying the histochemical
method of staining with silver.
In 15 pulmonary carcinomas – other than those with small
cells (NSCLC) we also examined the expression of epidermal
growth factor receptor (EGFR).
Quantification of Ki-67 immunoreaction
The IHC marking of the sections with MIB-1 antibody identified a staining pattern limited to the nucleus. We assessed Ki-67
immunoreactivity by evaluating tumor cells from evenly colored
areas, avoiding necrosis and hemorrhagic foci, as well as areasof inflammation. We considered as positive any detectable
nuclear staining (dotted or diffuse).
For the quantification of Ki-67 immunoreaction we determined the percent of reactive cells. Given the heterogeneity instaining distribution and intensity, to avoid subjectivity and difficulties in assessing the results, we used a semi-quantitative score(corresponding to staining intensity and the percent of reactive
nuclei), that allowed the classification of Ki-67 immunoreactivity
in three score groups:•
Low score (< 25% of tumor cells stained),•
Intermediate score (26-75% of tumor cells stained),•
High score (76-100% of tumor cells stained).
The marking of sections using PC10 antibody identified astaining pattern confined to the nucleus.
Silver staining of sections allowed visualization AgNOR likesome black or dark brown “drops”, distinct or grouped, with nucleolar or/and nuclear localization. For the numerical assessment of
NOR we counted 100 consecutive nuclei, calculating the mean
AgNOR per nucleus for each case, and for the measurement of
maximum diameter of NOR we used the ocular micrometer.
We interpreted as p53 positive reaction the distinct, homogenous or granular nuclear staining.
Positive EGFR immunoreaction was indicated by the darkbrown staining of cell membrane and cytoplasm.
Statistical analysis was performed using the EpiInfo 6.04,
EpiInfo 3.2.2 and OpenEpi 2.3.1 programs and consisted in computing the frequency counts and percentages for the qualitative
variables. The comparision of the percentages and the means
was performed using the chi-square teste and unpaired t -test.
RESULTSIn the majority of cases examined we observed heteroge2011.21.2 (70)  Fiziologia - Physiology

neity of immunoreactions (concerning intensity of staining andnuclear marking) (Fig. 1) and a variety of marking types: isolated
nucleolar marking (delicate, dotted), nucleolar and nucleocytoplasmatic marking, intense and diffuse marking of the entire
nucleus and intense marking of the nucleus and cytoplasm.

Fig. 1. Ki-67 immunoreaction with intense and granular nuclear pattern ofstaining. ABC method, viz. AEC x 400.

We remarked the absence of Ki-67 immunoreaction on
the negative control section and in normal pulmonary tissue. In
normal surface pulmonary epithelium and in hyperplastic basal
cells we noted the presence of rare, dispersed positive nuclei.
While in the squamous metaplastic pulmonary epithelium
without atypia, MIB-1 immunostaining marked isolated reactive
nuclei, in atypical squamous or dysplastic metaplasia and in
intraepithelial carcinomas associated with invasive squamous
carcinomas, we remarked the presence of a large number of
Ki-67 positive nuclei, with anarchic distribution throughout theepithelium (Fig. 2). We observed positive reaction for Ki-67 (withrare reactive nuclei) in pulmonary glands and in the nuclei ofstoma lymphocytes, and in a small number of cases we noted aclear and consistent staining of elastic fibers from alveolar septa
and vascular walls.

Fig.2. Intraepithelial carcinoma with Ki-67 positive nuclei disperssed
throughout the entire thickness of the epithelium. ABC method, viz. AEC x
400.

23

We identified Ki-67 immunoreactivity in 53 of the 62 primary pulmonary carcinomas analyzed (85.4%), the positive
reaction being characterized by a nuclear immunostaining
with variable intensity from case to case. The percent ofreactive nuclei varied widely, differing with the histologicalgrade. While typical carcinoids and bronchioalveolar carcinomas evaluated presented low values of the proliferative
activity, with rare Ki-67 immunoreactive cells (LI Ki-67 < 5%),small cell carcinomas presented the highest growing rate,
the majority of cases (10 of 12 cases; 83.3%) presenting
intermediate of high immunoreactivity, with over 25% Ki-67
reactive nuclei (Table I) and intense marking of the whole
nucleus (Fig. 3).

heterogeneity of marking in adenocarcinomas (Fig. 5) and insquamous cell carcinomas (Fig. 6).

Fig.5. Acinar pulmonary carcinoma. Positive Ki-67
immunoreaction with isolated Ki-67 reactive nuclei.
ABC method, viz. AEC x 400.

Fig.3. Small cell pulmonary carcinoma with high Ki-67 score (>75% reactive
tumor cells). ABC method, viz. AEC x 400.

The 7 undifferentiated big cell carcinomas, positive for
Ki-67, were characterized by an evident heterogeneity of Ki-67
expression, with variations of staining intensity, but especiallyof the types of marking (Fig. 4).

Fig.4. Undifferentiated big cell carcinoma. Heterogeneous Ki-67 expression
with variations of marking types. ABC method, viz. AEC x 400.

We found an intense proliferative activity and a similar
24

Fig.6. Squamous cell carcinoma. Positive Ki-67 immunoreactionin the nuclei of tumor cells.
ABC method, viz. AEC x 200.

Ki-67 was expressed by primary pulmonary tumors, butalso by corresponding metastases, hepatic metastatic lesions,metastases from lymphatic nodules and submandibular salivary
glands, being characterized by an intermediate or high proliferative activity.
We grouped pulmonary carcinomas with positive Ki-67
immunoreaction based on histological type and tumor differentiation degree. Using cellularity as criterion, we divided the studied
tumors in three groups; the cellularity of tumors was assessed ona scale from 1 to 3, the third degree having the highest degreeof cellularity.
Analyzing the relationship between the reminded morphological parameters and tumor proliferative activity, we observed asignificant relationship between cellularity (C), tumor histologicaldegree (G) and growth rate appreciated through Ki-67 score(Table I).

Fiziologia - Physiology  2011.21.2 (70)Table I. Ki-67 positivity rate correlated with cellularity and histological grade.

Variable (n) Ki-67
negativen (%)

Histological
type
0
SCLC (12)8 (17%)NSCLC (47) 1 (33%)
Carcinoid (3)Cellularity(C)
4 (26.6%)
C1 (15)
3 (12%)C2 (25)2 (9%)
C3 (22)
Histologicalgrade (G)G2 (17)G3 (45)
Number of
cases

Ki-67
positiven (%)

Ki-67 score groups
Low
Inter(<25%) mediateand high(25100%)12 (100%) 2 (16.6%) 10
39 (83%) 9 (48.7%) (83.3%)
2 (66%)
1 (50%) 20 (51%)
1 (50%)
11 (73.3%) 9 (81.8%) 2
22 (88%) 7 (31.8%) (18.1%)
20 (90.9%) 6 (30%) 15(68.1%)
14 (70%)7 (41.1%) 10 (58.8%) 5 (50%)
2 (4.1%) 43 (95.5%) 17(39.5%)
9 (14.5)
53 (85.4%) 22(41.5%)

5 (50%)
26(60.4%)
31(58.4%)

n = number of cases; SCLC = small cell lung carcinomas; NSCLC = non-small
cell lung carcinomas

The percent of Ki-67 positive cells correlated with the lowest
differentiation degree (G3) (p<0.001; ES) and the third degree ofcellularity (C3) was seen more frequently in small cell carcinomas
and in dedifferentiated areas of pulmonary carcinomas, otherthan those with small cells.
Among the patients with G2 histological grade of thetumor, 7(41.1%) were Ki-67 negative and 10 (58.8%) Ki-67
positive (p=0.672875; NS), while 2 (4.1%) G3 patients wereKi-67 negative and 43 (95.5%) were positive for this antigen(p<0.001; ES).
We analyzed the possible prognostic significance of Ki-67
expression on subgroups of patients that were classified basedon lymphatic nodules and metastasis potential (Table II).
Table II. The relationship between Ki-67 expression
and the evolutive character of tumors
Ki-67 immunoreaction (n)Positive
lymph
nodes

Invasionof pleura± thoracicwall
1 (11.1%)

Local
relapses

Ki-67 nega- 4 (44.4%)
1 (11.1%)
tive (9)Ki-67 posi- 21 (39.6%) 11 (20.7%) 6 (11.3%)
tive (53)pp=0.924448 p=0.825267 p=0.581481
(NS)
(NS)
(NS)
NS = not significant

Distant metastases
1 (11.1%)
13 (24.5%)
p=0.646270(NS)

In the group of the 53 carcinomas expressing Ki-67 nuclear
antigen we found implication of lymph nodes in 21 (39.6%) cases,
invasion of pleura ± extension into the thoracic wall in 11 (20.7%)
cases, local relapses in 6 (11.3%) cases and distant metastasis
in 13 (24.5%) cases; all these tumors were characterized by an2011.21.2 (70)  Fiziologia - Physiology

unfavorable evolution, resulting in 14 deaths within 21 months
from diagnosis.
We did not observe a statistically significant relationship between Ki-67 expression and lymph node involvement(p=0.924468; NS), invasion of pleura and/or thoracic wall
(p=0.825267; NS), local relapses (p=0.581481; NS) and distantmetastases (p=0.646270; NS).
We analyzed tumor proliferative rate (appreciated by Ki-67
score) in relation to p53 immunoreactivity (Table III).
Table III. Ki-67 immunoreaction correlated with p53 expressionin pulmonary carcinomas
Ki-67 score p53 immunoreaction
p53 negap
p53 posip
tive (n=30)
tive (n=32)
Ki-67 nega- 7 (23.35%) p=0.003431 3 (9.3%)
p<0.001
tive (n=9)
23 (76.7%) (VS)29 (90.6%) (ES)Ki-67 positive (n=53)
Low score 14 (60.8%) p=0.121395 8 (27.5%)
p=0.000465(<25%
9 (39.2%)(NS)
21 (72.4%) (ES)reactivenuclei)Intermediate andhigh score(>25%
reactivenuclei)VS = very significant; NS = not significant; ES = extremely significant

29 (90.6%) of the 32 carcinomas p53 positive presented
cells expressing Ki-67 nuclear antigen (p=0.001; ES), 21 cases(72.4%) having an intermediate and high Ki-67 score (Table III),with more than 25% reactive nuclei) ( p=0.000465; ES)In the subgroup of p53 negative tumors we noted thepresence of a large number of cases (23 cases; 76.6%) that
expressed positive immunoreaction for Ki-67 (p=0.003431; VS),significantly higher than the number of patients p53-, Ki67-; in
14 (60.8%) of these cases the antibody used (MIB-1) stained <25% of tumor cells.
In the p53 positive group, the number of patients with intermediate and high Ki-67 score is significantly higher than thenumber of patients with low score (p=0.000465; ES), while in thep53 negative patients, the difference between intermediate/highand low Ki-67 score was not significant (p=0.121; NS).
Among p53 negative patients, the number of Ki-67 positive tumors is significantly higher than Ki-67 negative tumors(p=0.0034; ES).
To appreciate the significance of Ki-67-EGFR co-expression,
we followed a possible relation between immunohistochemicallydetermined growth fraction and EGFR expression. We identified
positive Ki-67 immunoreaction especially in stage III and IV
tumors (9 cases); similarly, EGFR expression was proved to be
more frequently positive in advanced tumor stage than in earlystage, thus demonstrating a relationship between these markers
and invasive characteristics of the tumor.
The majority of T3 and T4 tumors (7 of 9 cases; 77.7%)25

presented high values of proliferative activity (Ki-67 score>25%) and positive EGFR immunoreactions (with a mean of
EGFR absolute staining score of 3), as compared with tumors
presenting less invasive features. Also, involvement of lymph
nodes and distant metastasis were more frequently observedin Ki-67+ EGFR+ cases (5 cases).
For a better definition of the prognostic roleof Ki-67 – EGFR
co-expression, we analyzed these two parameters after classifying tumors into 4 groups (based on immunoreaction positivity)(Table IV). group 1 included Ki-67- EGFR- tumors; group 2comprised Ki-67- EGFR+; in the 3rd group we included tumors
with Ki-67 positive (low score; Ki-67 <25%) and EGFR positive
immunoreactions; in the 4th group, EGFR positive tumors withintermediated and high Ki-67 score (Ki-67 >25%).
Table IV. Prognostic significance of Ki-67 – EGFR co-expression
Tumor
group

Ki-67EGFR-

Ki-67EGFR+
3

Ki67<25%
EGFR+
3

Ki67>25%
EGFR+
8Number ofpatients
Survival rate(months)

1
40

11

9

7

The results obtained show the highest survival rate (of 40
months), but one that was not attained at the last visit, in thecase of a stage II patient with a Ki-67- EGFR- tumor; relative tothis aspect, the analysis of survival rate in relation with Ki-67 and
EGFR expression suggests that positive Ki-67 immunoreaction (>25%) and simultaneous over-expression of EGFR anticipates agroup of pulmonary tumors with poor prognosis and the tendency
towards a shorter survival.
Analyzing the relationship between Ki-67 and PCNA positivity rate (Table V), we observed a higher percentage of tumors
with intermediate and high PCNA score (39 cases; 73.5%), as
compared with tumors positive for Ki-67 nuclear antigen fromthe same score groups (31 cases; 58.4%), but statisticallyinsignificant (p=0.074989).
Table V. Ki-67 and PCNA positivity rate
Ki-67
Nr. of
and
cases
PCNA
immunoreaction

Ki-67
positive
PCNA
positive

53
53

Score groups
Low
p
score
(<25%)

Interpmediateand highscore(25100%)22
p=0.215815 31
p=0.074989(41.5%) (NS)(58.4%) (NS)14
39
(26.4%)(73.5%)
NS = not significant

In the attempt of defining the relationship between thenumber AgNOR and immunohistochemical markers of proliferative activity, we compared the percentage of Ki-67 positive
and PCNA positive cells with mean AgNOR. In this sense we26

grouped pulmonary tumors according to Ki-67 and PCNA scores(Table VI), calculating the mean value of AgNOR/nucleus foreach score group.
Table VI. The relationship between mean number of AgNOR/nucleus and IHC
markers of proliferative activity (Ki-67 and PCNA)
Ki-67 (MIB-1) Score groups
Low (<25%)
Mean AgNOR/ 3.19
nucleus
PCNA (PC10) (<25%)
Mean AgNOR/ 3.04
nucleusIntermediate(26-75%)
3.5High (>75%)(26-75%)3.24(>75%)
3.41

3.57

Although the differences are not significant, the results weobtained show a tendency towards a higher mean number of
NORs, directly related to an immunoreactivity increase for Ki-67
antigen and PCNA, suggesting a weak correlation between mean
AgNOR and tumor growth rate appreciated by immunostaining
with MIB-1 and PC10.
DISCUSSION
In the intent of appreciating the prognosis of patients withpulmonary cancer, we investigated several parameters related
to aggressiveness of malignant growth, cellularity, positivity rate
for Ki-67 and PCNA. For the evaluation of proliferative activity weused, in 62 primary pulmonary tumors, the following methods: (1)determining the percent of immunoreactive cells for Ki-67 antigen
and PCNA and classifying Ki-67 and PCNA immunoreactivity in
3 score groups; so, we evaluated the proliferative activity shown
through MIB-1 antibody and PCNA staining; (2) assessing of
NORs using the histochemical method of staining with colloidal
Ag and (3) comparing Ki-67 score and the fraction of PCNA
positive cells with mean number of AgNOR per nucleus.
MIB-1 recognizes a nuclear antigen that is associated withall phases of cell cycle, except G0. Its presence in cells from thecell cycle and its absence from G0 cells allows the determinationof proliferation coefficient, the result being expressed in percent
of stained cells – so, the percent of Ki-67 positive cells could beconsidered as measurement of growth fraction of the studied
cell population (Seigneurin DG, 1991).
Studying the expression of Ki-67 antigen in normal andsquamous metaplastic bronchial epithelium without atypia,
we identified rare dispersed reactive nuclei, the majority being
situated in the basal layer. But, we observed a large number ofreactive nuclei throughout all the thickness of the epithelium inatypical squamous metaplasia and intraepithelial carcinomas
associated with invasive squamous tumors, in accord with Pendleton’s observations (1993) about Ki-67 expression in bronchialsquamous metaplasia.
We observed positive immunoreactions and heterogeneityof Ki-67 expression in 53 (85.4%) of the 62 primary pulmonary
tumors immunostained with MIB-1. IHC detection of Ki-67 antigen expression combined with other clinical-morphological andbiological parameters proved to have a real value in appreciating
the prognosis of patients with pulmonary cancer.
Fiziologia - Physiology  2011.21.2 (70)A significant relationship can be noted between Ki-67 staining and other prognostic traces; so, the percent of Ki-67 positive
cells (Ki-67 score) appears different through the various histological subtypes of pulmonary cancer, according to the stage anddegree of tumor differentiation, and thus confirming its prognostic
validity. Correlating Ki-67 score groups with histological types of
pulmonary cancer, we observed that most small cell carcinomas(83.3%) show >25% Ki-67 reactive nuclei, with an intermediate orhigh Ki-67 score, while only 51.2% of non-small cell carcinomas
are in the same score groups.
Typical carcinoids and bronchioloalveolar carcinomas presented a Ki-67 staining index <5% (low Ki-67 score), values thatare much lower than in small cell carcinomas and conventionalpulmonary adenocarcinomas; these differences, similar to thoseobserved by Kitamura (1995) and Przygodzki (1996), seem tobe related to the degree of atypia of the lesions, indicating theproliferation of a limited number of cells.
The data from literature referring to the prognostic valueof Ki-67 staining index are few, and the relationship between
Ki-67 positivity rate and prognosis is still unclear (Hashimoto K,
2004). Bouzubar et al. (1989) sustain that tumors that stain over
20% with Ki-67 have a high risk of relapse; Wintzer et al. (1991)
reported a more severe prognosis in patients with Ki-67>16%
values, while Sabin (1991) noted a lower probability of survival
at 5 years of patients with Ki-67>13% values (Veronesse SM,1993).
In the study of Thaler (1987), detection of Ki-67 antigen
proved to be superior to the determination method of differentiation histological grade or of mitotic index, and he considers thatno matter the histological type, all tumors with over 5% Ki-67
positive cells have an adverse clinical course (Veronesse SM,1993).
The results of Nakano and Oka (1993) sustain that highproliferative activity is correlated with high tumor malignity, withrelapses and high metastasis rate. The higher the proliferative
activity of a tumor, the higher its malignant potential, and survival(from the moment of diagnosis) is reduced (Pugsley JM, 2002;
Skov BG, 2010).
Our results point out a suggestive relationship between
Ki-67 immunostaining, advanced disease stage (III and IV) andhigh cellularity (C3), histological type of small cell carcinoma andpoor tumor differentiation (in accord with similar observations
reported by Kawai T, 1994); we found these parameters to beclosely correlated between them, as well as with prognosis, whichsuggests the possibility of their use as factors in appreciating
clinical behavior. Involvement of lymph nodes and high potential of metastasis correlated well with tumors expressing Ki-67
nuclear antigen, characterizing a group of neoplasms with rapid
evolution and death in the first two years from diagnosis.
Given the allegation of the relation between gene increaseor overexpression and tumor proliferative activity (Ştefănescu
DT, 1996), we analyzed the relationship between growth rate
and p53 immunoreactivity, and on the other hand we followed
the relation between proliferative activity and EGFR positivity
rate (estimated by calculating Ki-67 score and EGFR absolute2011.21.2 (70)  Fiziologia - Physiology

staining score).
We found a p53 overexpression in 29 (90.6%) of the 32
Ki-67 positive pulmonary tumors and in 21 (72.4%) of tumors
with intermediate or high Ki-67 score (with >25% reactive nuclei), these results suggesting the prevalence of p53 positive
immunoreactions in poorly differentiated pulmonary tumors withhigh proliferation rate.
The observations concerning the relationship between p53
and Ki-67 antigen expression in pulmonary cancer are controversial (Maddau C, 2006). In this respect, evaluating the prognosis
significance of Ki-67 and p53 expression in resection non-small
cell pulmonary carcinomas, Scagliotti (1994) does not remark
any significant correlation; but, the author observes a relatively
high risk of recurrence and a lower rate of survival of patientswith >25% Ki-67 positive tumor cells, our results confirming
these last observations.
In the group of tumors with positive Ki-67 immunoreaction
we noted a high number of p53 negative cases; these results
probably suggest that p53 gene mutations are not the only
mechanisms involved in acquiring the proliferative potential.
Little is known about a possible relationship between
growth fraction immunohistochemically determined and EGFR
expression in pulmonary carcinoma. In 15 of the 62 carcinomas
immunohistochemically studied we analyzed EGFR expression
and its relationship with proliferative activity estimated throughKi-67 score. We remarked that Ki-67 positivity is in close relation
with EGFR positive immunoreactions; all Ki-67 positive tumors
expressed EGFR, while only 3 Ki-67 negative tumors proved
positive EGFR reaction. So, it seems that high expression of
Ki-67 and EGFR could be related with tumor evolution.
EGFR expression in high proliferative tumors (Ki-67 scoreof 75-100%) diagnosed in an advanced stage of the disease,with positive lymph nodes, suggest that simultaneously positive
expression of Ki-67 antigen and EGFR (Ki-67+ EGFR+) reflects
the invasive character of pulmonary cancer, being considered
indicators of malignancy. These results clearly indicate a tight
relationship between growth rate (Ki-67>25%), high EGFR expression and clinical-pathological features of an invasive tumor
behavior, and Ki-67+ EGFR+ co-expression is an indicator orpoor prognosis, high invasion and metastasis potential in patientswith pulmonary cancer.
The observations from literature regarding the relationshipbetween PCNA and Ki-67 antigen expression in pulmonary cancer are rather controversial (Koohdani F, 2009). While Rowlands(1994) notes lack of a relationship between Ki-67 and PCNA
score, in our study PCNA immunoreaction corresponded rather
well to Ki-67 growth fraction; but, we tracked a higher number oftumors with intermediate and high PCNA score, as compared to
Ki-67 positive tumors from the same score groups.
The relationship between positive PCNA immunoreactions,
Ki-67 positivity rate and metastasis potential (involvement oflymph nodes and distant metastasis) suggest the predictive
value of these factors linked to proliferation, in pulmonary cancer. We can say that tumor cells with high proliferative activity
anticipate a higher malignancy of the disease, consisting in a27

higher frequency of relapses and metastases, confirming theobservations of Kawai and Suzuki (1994).
If, as suggested, AgNOR plays a role as cell proliferation
markers (Seigneurin DG, 1991; Crocker J, 1987), then correlations with mitotic activity and Ki-67 and PCNA immunoreactivity
are to be expected. In this respect, to determine how much thenumber of AgNOR is related to proliferative activity, we studied
the relationship between the mean number of AgNOR per
nucleus and tumor growth rate estimated through assessmentof Ki-67 and PCNA score.
We observed a tendency of increasing mean AgNOR
value in parallel with growing of tumor cell proliferative activity(appreciated through Ki-67 and PCNA immunostaining). The
weak correlation between the number of AgNOR and immunohistochemical markers for proliferative activity, also found in
the study of Rowlands, pleads for the widely accepted idea thatAgNOR mean number can be a valuable parameter, useful in
predicting tumor malignant potential (Fushiki S, 1996; Cornianu
M, 1999).
The results obtained, demonstrating the increase of AgNOR
mean number parallel with the increase of Ki-67 antigen and
PCNA immunoreactivity, are in accord with the observations ofother authors, pointing out that these parameters can reflect thestate of cell proliferation (Aleen JP, 1992); on the other hand,
AgNOR mean number seems to reflect closely the rate of tumor
growth and can be used as index of proliferative activity (Ogura
S, 1989; Cornianu M, 1999).
CONCLUSIONS•
We identified cells that express Ki-67 antigen (MIB-1
positive cells) in 53 (85.4%) from the group of 62 pulmonary
carcinomas studied immunohistochemically. After correlating thegroups of Ki-67 score with other prognostic variables, we noteda relationship between Ki-67 immunostaining, advanced stageof the disease (III and IV) and high cellularity (C3), histologicaltype of small cell carcinoma and poor tumor differentiation. These
parameters are intimately correlated between them, as well aswith prognosis, suggesting the possibility of their use as factors
in appreciating proliferative status and clinical behavior.
•
Lymph node involvement and high metastasis potential
in patients with Ki-67 positive tumors characterized a groupof carcinomas with short evolution and death because of thedisease in the first two years from diagnosis.
•
29 (90.6%) of the 32 p53 positive carcinomas presented cells expressing Ki-67 antigen, 21 cases (72.4%) having an intermediate and high Ki-67 score (with >25% reactive
nuclei); the results obtained show the prevalence of positive p53
immunoreaction in poorly differentiated pulmonary tumors witha high proliferation rate.
•
All Ki-67 positive tumors expressed EGFR. EGFR
overexpression in highly proliferative tumors (with >75% Ki-67
reactive nuclei) diagnosed in the advanced stage of the disease,with positive lymph nodes, suggest that simultaneous expression Ki-67+ EGFR+ is an indicator of poor prognosis, invasion
and increased metastasis potential in patients with pulmonary
28

cancer.
•
PCNA immunoreaction corresponded rather well to
Ki-67 growth fraction; the relationship between PCNA immunostaining, Ki-67 positivity rate and metastasis potential showsthat high proliferative activity anticipates a higher malignancy
of the disease, consisting in a higher frequency of relapses andmetastases.
The weak correlation between mean AgNOR per nucleus
and IHC markers for proliferative activity, suggested by thetendency of increase of the mean AgNOR number, proportional with the increase of immunoreactivity for Ki-67 antigen and
PCNA, sustains that these parameters can reflect the state ofcell proliferation.
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EXPRESIA SI SEMNIFICATIA ANTIGENULUI KI-67 IN CANCERELEPULMONARE. RELATIA DINTRE KI-67 SI ALTI FACTORI DE PROGNOSTICREZUMATAm apreciat activitatea proliferativă în 62 de carcinoame pulmonare primitive, prin: determinarea procentului de celule Ki-67 imunoreactive (marcare cu anticorpul MIB-1), prin compararea scorului Ki-67 cu fracţia de celule PCNA pozitive (evidenţiate cu anticorpul
PC10), şi prin evaluarea regiunilor de organizare nucleolară (NORs) prin metoda histochimică de colorare cu argint coloidal şi
compararea scorului Ki-67 şi a fracţiei de celule PCNA pozitive cu numărul mediu al AgNOR per nucleu.
Am urmărit relaţia dintre rata de creştere Ki-67, imunoreactivitatea p53 şi nivelul receptorului pentru factorul epidermal de creştere
(EGFR), prin colorarea secţiunilor cu anti-p53 (DO-7) şi anti-EGFR (31G7); sectiunile tisulare fixate in formol si incluse la parafina
au fost colorate imunohistochimic prin metoda Avidin Biotin Complex (ABC).
Rezultatele relevă: (1) o rată de pozitivare Ki-67 corelată semnificativ statistic cu gradul histologic (G3) al tumorii (p<0,001; ES) şicelularitatea crescută (C3); (2) o relaţie nesemnificativă statistic între expresia Ki-67 şi implicarea limfonodulilor (p=0.924448; NS),invazia pleurei şi/sau a peretelui toracic (p=0.825267; NS), recidivele locale (p=0.581481; NS) şi metastazele la distanţă (p=0.646270;
NS); (3) prevalenţa expresiei p53 pozitive in tumorile pulmonare slab diferenţiate, cu rată crescută de proliferare (p<0.001;ES); (4)nivelul crescut al expresiei EGFR în tumorile înalt proliferative, diagnosticate în stadiul avansat de boală, ce reflectă caracterulinvaziv şi potenţialul crescut de metastazare al carcinoamelor pulmonare Ki-67+ EGFR+; (5) o slabă corelaţie între media AgNOR
per nucleu şi markerii IHC ai activităţii proliferative.
Rata de pozitivare Ki-67 şi PCNA corelată cu celularitatea tumorii, aduc alături de stadiul clinic şi diferenţierea histologică un plus
de informaţie utilă în anticiparea evoluţiei şi în prognozarea cancerului pulmonar.
Cuvinte cheie: carcinom pulmonar, Ki-67, PCNA, imunohistochimie

2011.21.2 (70)  Fiziologia - Physiology

29

DETECTION OF ANTI-p80 AUTOANTIBODIES IN PATIENTSWITH ALZHEIMER’S DISEASE
MANOLE COJOCARU1, INIMIOARA MIHAELA COJOCARU2
1“Titu Maiorescu” University Faculty of Medicine, Department of Physiology, Bucharest,2”Carol Davila” University of Medicine and Pharmacy, Department of Neurology, Colentina Hospital, Bucharest, Romania

ABSTRACTThe protein p80 coilin is a nuclear autoantigen that strongly accumulates in Cajal bodies and is considered a marker for Cajal bodies which
are ubiquitous nuclear structures frequently observed in close proximity to nucleoli. The protein p80 coilin is a target for autoantibodies
identified in the sera of patients with diverse autoimmune features. Several clinical studies have investigated the distribution of anti-p80
coilin antibodies in various diseases. However, the clinical significance of anti-p80 coilin antibodies is not fully understood.
The objective of this study was to evaluate the anti-p80 coilin autoantibodies in serum of patients with Alzheimer’s disease and toanalyze the link between p80 coilin autoantibodies outcome of the disease.
Serum samples from 47 patients with Alzheimer’s disease 27 women and 20 men (mean age 70.43±10.82 years, range 40-89 years)
as compared to 47 control subjects, 25 women and 22 men (mean age 70.17±10.64 years, range 40-89 years). Patients with evidence
of infectious or inflammatory disease were excluded. For screening of anti-p80 coilin antibodies, indirect immunofluorescence was
performed with HEp-2 cell slides.
Anti-p80-coilin antibodies produce a unique pattern of immunofluorescence staining called nuclear dots. Five patients with Alzheimer’s
disease showed the nuclear dot pattern by immunofluorescence. Our data indicate that 10% were positive for anti-p80-coilin antibody.
In conclusion, immunological disturbances seem to be a common feature of patients with Alzheimer’s disease in the form of local
brain tissue inflammatory reactions and autoagressive processes directed against the brain cells. Increased plasma levels of theinflammatory marker IL-6 are associated with worse of disease. Interleukin-6 assists in risk stratification of patients with Alzheimer’s
disease. Autoantibody to p-80 coilin may be useful for the elucidation of the structure and function of the coiled body.
Key words: Alzheimer’s disease (AD), anti-p80 coilin autoantibodies, chronic neuroinflammation

INTRODUCTION
The cause of Alzheimer’s disease remains unknown. There isa growing of body evidence which supports the hypothesis of faulty
immune regulation and autoimmunity or inflammatory processes as
viable mechanisms of the pathogenesis of the disease. There are notpublications reporting anti-p80 autoantibodies in the serum of patientswith Alzheimer’s disease. Several clinical studies have investigatedthe distribution of anti-p80 coilin antibodies in various diseases. P80coilin, a nuclear autoantigen with a molecular weight of 80 kda, is
one of several proteins localized in structures called “coiled bodies”.
Historically, these nuclear structures were first described by the cellbiologist Ramon y Cajal and subsequently identified as “Cajal bodies”.
p80 coilin is a nuclear autoantigen that strongly accumulates in Cajalbodies (CB) and is considered a marker for CBs. No clear clinicalfeatures have been associated with anti-p80 autoantibodies, andthe epitopes recognized by these antibodies are entirely unknown.
Anti-p80 coilin antibodies are present in some patients with clinicaldisease but with no discernible pattern (1-3)
MATERIALS AND METHODS
47 serum samples obtained from AD patients were analyzed
for anti-p80 coilin antibodies by indirect immunofluorescence. All AD
patients fulfilled the following criteria: 1) slow progressive decline
of intellectual function; 2) a score on the clinical dementia rating
scale more than 0.5; 3) a score on the short portable mental statusquestionnaire (SPMSQ) of less than 20; 4) a score of 7 or less on the
Hachinski-scale; and 5) no evidence for abnormalities on CT-scanother than cerebral atrophy, and no evidence for focal dysfunctionin the EEG. Exclusion criteria were: infections, neoplasias, surgicalinterventions, autoimmune disorders, concurrent major renal or
hepatic disorders, major trauma in previous month. Informed consent
was obtained from all patients and controls, and approval was also
obtained from the Ethics Committee. Sera were diluted at 1:40 in
PBS. For screening of anti-p80 coilin antibodies, indirect immunofluorescence (IIF) was performed with HEp-2 cell slides.
We used Fisher’s exact test for the analysis of autoantibody
frequency, and unpaired t-test. P values of less than 0.05 wereconsidered significant.
RESULTS
Anti-p80 coilin antibody produces a unique pattern of
immunofluorescence staining called nuclear dots characterized
by the presence of up to six discrete nuclear bodies in interphasecell nuclei. Our data indicate that 10% were positive for anti-p80
coilin antibody. Autoantibody to p-80 coilin may be useful for the
elucidation of the structure and function of the coiled body. Five
patients with Alzheimer’s disease showed the nuclear dot patternby immunofluorescence, and reacted by immunoblotting with 80kDa protein in a nuclear extract from HeLa cells. Anti-p80 coilinshould be considered natural autoantibodies present in the seraof healthy individuals. The decreased prevalence of anti-p80 coilin
autoantibodies in the serum of older individuals suggests thattiters may decrease with age. Longitudinal studies are required
to clarify this. Anti-p80 coilin in patients with Alzheimer’s disease

Received May 10th, 2011. Accepted May 20th, 2011. Address for correspondence: Assoc. Prof. Manole Cojocaru, MD, PhD, “Titu Maiorescu” University, Faculty of
Medicine, Bucharest, 5 Thomas Masaryk Street 020983, Sector 2, Bucharest, Romania, e-mail: manole_cojocar@yahoo.com

30

Fiziologia - Physiology  2011.21.2 (70)

may reflect the specific impairment of helper T cell activity for B
cells that produce anti-p80 coilin.

but rarely in patients with connective tissue disease (CDT).
The clinical significance of many other antinuclear antibodies(ANAs) is not understood. One such ANA is directed against
p80 coilin is a specific marker of Cajal bodies (CBs) (7,8).
We find these results very promising for the considerationof future treatment of AD patients. The preliminary results need
be confirmed by controlled studies.
CONCLUSION
Anti-p80 colin autoantibodies are found during ANA
screening with HEp-2 cells. We conclude that the presence ofanti-p80-coilin autoantibodies is not associated with any particular clinical syndromes nor is it diagnostic of any conditions.

Fig.1. The coiled body pattern by indirect immunofluorescence on HEp-2 cell
substrate. Discrete round bodies (zero to 6 per cell) randomly distributed in the
nucleoplasm of interphase cells (often located in the vicinity of the nucleolus)

DISCUSSION
The coiled bodies appear as a tangle of coiled, electron-dense
threads roughly 0.5 mm in diameter. The only epitope reported
to date that can be used as an unambiguous marker for coiled
bodies is a protein called p80 coilin. Although there is certainly apool of this protein localized diffusely throughout the nucleoplasm,
antibodies against coilin typically stain a few discrete foci per
nucleus. Coiled bodies are highly enriched in several classes of
snRNPs, nucleolar 9 e.g., fibrillarin, topoisomerase I) and cell-cycle
conrol proteins (e.g., cyclin E, cyclin H), as well as several basaltranscription factors. Of note, coiled bodies play a major role indirecting intranuclear snRNA sorting and might even be an obligate
waystation in the biogenesis pathways of snRNPs and snoRNPs.
The protein p80 coilin is a target for autoantibodies identified in thesera of patients with diverse autoimmune features (4-6).
Coilin autoantibodies are rarely detected. This autoantibody
is sometimes found in patients with some allergic diseases

REFERENCES

1. Fujimoto M, Kikuchi K., Tamaki T., Yazawa N., KuboM., Ihn H., et al.
Distribution of anti-p80-coilin autoantibodies in collagen diseases andvarious skin diseases. BJD, 1997; 137: 916-20.
2. Matera AG. Nuclear bodies: multifaceted subdomains of the interchromatin space. Trends Cell Biol., 1999; 9: 302-309.
3. Andrade LE, Chan EK, Raska I, Peebles CL, Roos G, Tan EM. Human autoantobody to a novel protein of the nuclear coiled body:immunological characterization and cDNA cloning of p80-coilin. J Exp
Med. 1991; 173: 1407-19.
4. Hiepe F. The particle of intracellular autoantigens In: Autoantigens
and autoantibodies: diagnostic tools and clues to understanding autoimmunity. K. Conrad, R-L. Humbel, M. Meurer, Y. Shoenfeld, E. M. Tan,
Pabst Science Publisher. 2000; p. 762.
5. Blank M, Gershwin E. Autoimmunity: From the mosaic to the kaleidoscope. Journal of Autoimmunity, 2008; 30: 1-4.
6. Tsonis IA, Avrameas S, Haralampos M. Autoimmunity and pathophysiology. Journal of Autoimmunity, 2007; 29: 203-205.
7. Zelenay S, Fonts MFM, Fesel C, Demengeot J, Coutinho A. Physiopathology of natural autoantibodies: The case for regulation. Journalof Autoimmunity, 2007; 29: 229-235.
8. Nilsson BO, Skogh T, Ernerudh J, Johansson B, Löfgren S, Wikby
A, Dahle C. Antinuclear antibodies in the oldest – old women and men.
Journal of Autoimmunity, 2006; 27: 281-288.

DETECTIA AUTOANTICORPILOR ANTI-P80 LA PACIENTII CU ALZHEIMERREZUMAT
Proteina p80 coilin este un autoantigen nuclear, care se acumuleaza la nivelul corpilor Cajal, fiind considerata un marker al acestora,care sunt structuri nucleare ubicuitare, frecvent observate in apropierea nucleolilor. Proteina p80 coilin reprezinta tinta autoanticorpilor
identificati in serul pacientilor cu diferite caracteristici autoimmune. Multiple studii clinice au investigat distributia anticorpilor anti-p80
coilin in diferite afectiuni. Cu toate acestea, semnificatia clinica a anticorpilor anti-p80 coilin nu este pe deplin inteleasa.
Obiectivul acestui studiu a fost evaluarea autoanticorpilor anti p-80 coilin in serul pacientilor cu Alzheimer si analizarea legaturii
dintre acestia si evolutia bolii.
Au fost utilizate probe de ser de la 47 de pacienti cu Alzheimer, 27 de femei si 20 de barbati (varsta medie 70.43±10.82 ani, cu
varste cuprinse intre 40-89 ani) si comparate cu 47 subiecti de control, 25 femei si 22 barbati (varsta medie 70.17±10.64 ani, cu
varste cuprinse intre 40-89 ani). Au fost exclusi pacientii care au prezentat semne ale unei afectiuni inflamatorii sau infectionase.
Pentru screening-ul anticorpilor anti-p80 coilin, a fost utilizata metoda de imunofluorescenta indirecta, folosind lame Hep-2.
Anticorpii anti-p80-coilin produc un tip unic de imunofluorescenta, numit puncte nucleare. Cinci dintre pacientii cu Alzheimer auprezentat aceasta imunofluorescenta nucleara. Datele noastre indica faptul ca 10% dintre probele selectate au fost pozitive pentru
anticorpii anti-p80 colilin.
In concluzie, perturbarile imunologice par sa fie o caracteristica comuna la pacientii cu Alzheimer, sub forma reactiilor inflamatorii
locale cerebrale si procese auto-agresive orientate impotriva celulelor cerebrale. Cresterea plasmatica a nivelului interleukinei IL-6,
marker al procesului inflamator, este asociata cu un prognostic negativ in evoulutia bolii. IL-6 este implicata in stratificarea riscului lapacientii cu Alzheimer. Autoanticorpii pentru p-80 coilin ar putea fi utili pentru elucidarea structurii si functiei “coiled body”.
Cuvinte cheie: Alzheimer (AD), autoanticorpi anti-p80 coilin, neuroinflamatie cronica
2011.21.2 (70)  Fiziologia - Physiology

31

PREVALENCE OF HIGH RISK GENOTYPES OF HUMAN
PAPILLOMA VIRUS AMONG FERTILE WOMEN IN THE
WESTERN PART OF ROMANIAZUGRAVU ROXANA1,2, GHEORGHIU ELEONORA2, GHEORGHIU GEORGETA2,CRISTEA ANCA2, HOGEA ELENA1, DRAGOMIRESCU LILIANA1, LICKER MONICA1,MOLDOVAN ROXANA1
1
University of Medicine and Pharmacy “Victor Babes” Timisoara – Microbiology Department
2
Bioclinica SA Timisoara – Molecular Biology Department

ABSTRACT
Human Papilloma viruses (HPV) are widely distributed, producing epithelial and mucous tumors and are involved in malignant genital
pathogenesis. There are more than 100 known genotypes of HPV, of which approximately 40% infect genital mucosa.
During 01.03.2007 – 19.09.2009, Bioclinica Laboratories, tested 9245 women for detecting HPV infection. 38.27% were positive forat least one genotype, from which, 66.31% were high risk genotypes.
DNA extraction was automated performed (Magna Pure Roche) and genotyping by LINEAR ARRAY HPV Genotyping Test
(Roche).
From the high-risk one’s, type 16 was detected the most (20.68%), followed by genotypes 31 (13.89%), 51 (12.39%), 33 (8.75%), 66(7.96%), 18 (6.78%), 52 (5.87%), 58 (5.37%), 68 (3.70%), 45 (3.55%), 39 (3.08%), 56 (3.05%), 59 (2.85%) and 35 (2.06%).
Keywords: HPV, genotype, prevalence, high risk

INTRODUCTION
Human Papilloma viruses (HPV) are widely distributed,
producing epithelial and mucous tumors and are involved inmalignant genital pathogenesis (14). In Romania, cervical cancer
is up to 15% of total number of malignancies, being first as agenital cancer (aprox. 67% of genital cancers) and second place
as cancer death among women (1).
Cervical cancer has an overall good prognosis; survival at
5 years is 100% in phase 0, 91% in phase I, 83% in IIA phase,
66% in IIB, 45% in IIIA, 36% in IIIB, and 10-14% in phase IV.
Due to this, the main aim of cervical cancer treatment is acurative (local control), except phase IV, where the treatment
is palliative (12).
Epidemiological studies revealed that, the incidence of cervical cancer is considerably increased among women with a low
life status, early start of sexual life, sexual promiscuity, multiple
pregnancies and births, and among smokers.
Main etiology of cervical cancer and pre-neoplasic lesions
is due to human papilloma virus infection (8). The viruses are
small (45-55 nm), spherical, non-enveloped. HPV genome isformed from approximate 7600 base pairs. All supposed codifying
sequences (open reading frames - ORF) are arranged on one
DNA strand and all papillomaviruses have the same organization.
Various protein products are derived from these ORF’s.
Anogenital HPV subtypes are classified in “low risk”, forexample HPV6 and HPV11, which are associated with squamous
lesions, rarely causing cancer, and “high risk”, HPV16, HPV18,
associated with cervical intraepithelial neoplasia or squamous
lesions which can progress to cancer (2, 3, 9, 13). Experimental

studies showed that most cervical neoplasia, intraepithelial orinvasive one, are caused by an HPV infection. High risk DNAHPV can be found in 90% of human cervical cancers. Most of theinfections cannot be detected even with the slightest method of
DNA detection. Papanicolau test can detect some abnormalities
in the cervix. Both partners require examination and treatment,
to avoid reinfection; for relapses prevention, a permanent andprolonged medical surveillance is recommended.
Papillomaviruses do not grow in cell cultures. Thus, thelaboratory diagnose implies:
A. Microscopically examination of lesions
B. Electronic microscopy
C. Serology
D. Molecular biology techniques.
Papillomaviruses have tropism for squamous epithelial
tissue of skin and mucous membranes. At this level, the virus
is replicating and induces tissue proliferation, forming tumors.
Infection is by close contact (including sexual) through somelesions, even very small.
Viral replication and gene expression depends on differentiation degree of the infected epithelial
cell, causing persistent infections at the basal layer and active
infections in keratinocytes. Usually, the infection is localized
and spontaneously regressing. In some papillomaviruses, viralgenome can persist intracellular (viral DNA integration in hostcell), causing recurrences.
Clinical syndromes of HPV infection are:•Skin verruca•
Benign tumors of head and neck•
Genital condylomatosis

Received March 10th, 2011. Accepted May 20th, 2011. Address for correspondence: Zugravu Roxana, MD, I. Nemoianu Street No. 16, Timisoara 300011, Romania,
phone: +40745 18 27 28, e-mail: zurolx@yahoo.com

32

Fiziologia - Physiology  2011.21.2 (70)•
Cervical dysplasia and neoplasia•
Cervical intraepithelial neoplasia•
Bowen disease•
Invasive cervical carcinoma (4, 5, 6, 7)
Motivation for this study was represented by increasing
frequency of cervical cancer and the ability of ambulatory testingof circulating Human Pappiloma virus genotypes (responsible forthis type of cancer), combined with increased addressability of thefemale patients. Thus, the study reveals the frequency of circulating
genotypes of HPV among females, as well as persistency in time.
MATERIAL AND METHODS
The samples for this study were obtained from female
patients who addressed themselves at Bioclinica Laboratoryfor HPV-DNA detection, during 01.03.2007 – 19.09.2009. 9245
cervical samples were analyzed, from 8858 patients, age varying
from 12 to 73 years old.
From 8858 patients, 413 were
present again for re-testing, a good opportunity for surveillance ofvirus persistency. Cervical samples were collected in specialist’s
office, with the kit provided by the laboratory. Exfoliated cytology was used to obtain the samples, this being a non-invasive
collection method for screening in a normal gynecology check,collection is not based on the presence of a lesion. The sample’squality depends on collector’s type and the anatomical site, forwomen, cervical samples being the most reliable.
Age distribution among patients show an increasing trend
until 29 years of old (570 patients), after that, the addressability
decreases progressively (Figure 1).

DNA extraction was performed automatically (MagnaPure
Roche) and genotyping by LINEAR ARRAY HPV Genotyping Test
(Roche). LINEAR ARRAY HPV Genotyping Test is a qualitativein vitro assay for detection of HPV in clinical samples. The test
uses polymerase chain reaction for amplifying the target DNA,followed by hybridization, detecting 37 anogenital genotypes of
HPV (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54,
55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9),
81, 82 (MM4), 83 (MM7), 84 (MM8), IS39 and CP6108).
RESULTS AND DISCUSSIONS
From 9245 tested samples, 3538 were positive for at least
one genotype (38.27%) and 5107 were negative (61.73%)(Figure 2).

Fig.2. Distribution of positive and negative samples

Distribution of positive results versus negative results, revealed that at 21-25 age group, the positive samples were morethan negative’s, unlike other age groups, where the negative
samples were predominant (Table I).
Table I. Age distribution of positive and negative samples

Fig.1. Age distribution

2011.21.2 (70)  Fiziologia - Physiology

Identified DNA-HPV genotypes were 66.31% high-risk ones,
10.44% low-risk, and the rest, 23.25% - other HPV genotypes(Figure 3).
33

Fig.3. Genotype distribution among positive samples

High risk genotypes of HPV, detected in this study, arerepresented by: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66
and 68. From the high-risk one’s, type 16 was detected the most(20.68%), followed by genotypes 31 (13.89%), 51 (12.39%), 33(8.75%), 66 (7.96%), 18 (6.78%), 52 (5.87%), 58 (5.37%), 68(3.70%), 45 (3.55%), 39 (3.08%), 56 (3.05%), 59 (2.85%) and35 (2.06%) (Table II).
Table II. High risk genotypes distribution

Papilloma virus persistency at genital level is considered to
be a risk factor for developing a cervical neoplasia. From 8858
female patients included in the study, during 30 months, only one
patient was tested 5 times, 3 patients – 4 times, 25 – 3 times
and 384 – 2 times. The patient tested 5 times (23 years old)
was positive every time. In all 5 tests, genotype 58 persisted intime. The 3 patients tested 4 times, one was positive for every
test, one negative, and one was intermittent positive/negative
each time. 25 patients were tested 3 times. They had various
results regarding the presence or the absence of infection. Only
2 of them had negative results each time, and 8 were constantpositive. 384 patients were tested twice. 125 were negative inboth testing, 117- positive, 120 – eliminated the virus, and 22,
although a first test was negative, the second one showed an
infection. Distribution of persistent genotypes over time, revealed
34

that genotype 16 persisted for 4, 8, 12 and 16 months, followed
by genotype 66 – at 8, 12, 16 months.
All existent prophylaxis guides for cervical cancer, havepractical recommendations for splitting screening groups, thetime between testing and strategies to be followed for specialgroups of patients.
Optimal age for beginning of screening is different due tofactors considered by various authors:• From 25 years of old ;• From 21 years of old, or after 3 years from the sexual
life debut;• From 18 years of old because of the high frequency of
sexual activity at this age;
• From 30 years of old for new programs based on HPVDNA testing; from 25 years for existent programs (classicalcytology)
Now, there are no scientific arguments to establish the
optimal age for screening start.
The age from which the screening is no longer justified:• From 60 years;• From 65 if by this age a proper screening was performed,
the results were negative and the woman did not belong to any
risk groups;• From 70 years old if there are three successive negative
cytology results and no positive results in the last 10 years.
At the same time, testing is recommended for older women if
before they were not tested or there are no results from previous testing;• Patients more than 70 years old, DNA-HPV positive, must
continue testing in the recommended screening program;• From 65 – if the last two smears were negative• there are no limits in age for screening
Screening should be performed every 3-5 years, but no lessthan once every 3 years, if the procedure is based on classicalsmears, or every 2 years if the liquid based cytology is used.
The period can be prolonged to 2-3 years for patients above 30
years old, considering the associated risk factors and the result
from previous cytology exam. This recommendation is based onsensibility of cytology exam.
A female patient, initially HPV negative, can be infected,
becoming HPV positive, but the way to develop cervical cancer isdepending on the involved genotype, persistency in time, associated factors: age, immunity, co-infection with other genotypes,
life style, multiple births, smoking, use of contraceptive devices
etc. (10, 11, 15, 16)
CONCLUSIONS
Considering the complications which can occur from thisinfection, with severe repercussions, a management revisionof infections on fertile age is required, by approaching thisproblem from different pathways, as a diagnosis and treatment, and also as evaluating the knowledges, atitudes, behaviour for best corrective or preventive measures. It implies
immediat measures for primary and secondary prophylaxis.
Fiziologia - Physiology  2011.21.2 (70)

Some of those, more important are: education, mass screening, correct management of cases and partners, increasing
the quality and accesability of medical services.
REFERENCES

1. *** - Ghid de diagnostic si tratament al cancerului de col uterin, responsabil: Prof. Dr. Rodica Anghel, Presedinte Comisia de Oncologiea Ministerului Sănătătii, http://www.ms.ro/.
2. Abramova L, Parekh J, Irvin WP, Rice LW, Taylor JrPT et al. Sentinelnode biopsy in vulvar and vaginal melanoma: presentation of six cases
and a literature review. Ann Surg Oncol., 2002; 9 (9): 840-6.
3. Altekruse SF, Lacey Jr JV, Brinton LA, Gravitt PE, Silverberg SG,
et al. Comparison of human papillomavirus genotypes, sexual, andreproductive risk factors of cervical adenocarcinoma and squamouscell carcinoma: Northeastern United States. Am J Obstet Gynecol.,2003; 188: 657-63.
4. Arvas M. Early stage cervical cancers, in Ayan A, Gultekin M, Dursun
P, eds. 2009, Textbook of Gynecological Oncology, Gunes Publishing,Ankara, Turkey, pp 157-163.
5. Aull K, Friese K, Gisssmann L, et al. Recommendations for the
Diagnosis and Treatment of HPV Infections of Female Tract. Recommendations of European Society of Infectious Diseases in Obstetrics
and Gynecology, 2002 (ESIDOG), http:// www.esidog.com
6. Awen C, Hathway S, Eddy W. Efficacy of ThinPrep preparation of
cervical smears. Diagn Cytopathol., 1994; 11:337-43.

7. Bekkers R, Meijer C, Massurger L, et al. Effects of HPV detection in
population-based screening programmes for cervical cancer; a Dutch
moment. Gynecol Oncol., 2006; 100(3): 451-4.
8. Centers for Disease Control and Prevention. Sexually Transmitted
Diseases Treatment Guidelines, 2002.
9. Clavel C, Cucheroussset J, Lorenzato M, et al. Negative humanpapillomavirus testing in normal smears selects a population at low
risk for developing high-grade cervical lesions. Br J Cancer, 2004;
90(9): 1803-8.
10. Dubin G. HPV vaccine Adolescent Study Investigators Network.
Enhanced Immunogenicity of a Candidate Human Papillomavirus (HPV)
16/18 L1 Virus like Particle (VLP) Vaccine with Novel AS04 adjuvant in
Preteens/Adolescents. Interscience Conference on Antimicrobial Agentsand Chemotherapy (ICAAC), Washington, December 16-19, 2005.
11. European Cervical Cancer Screening Network. European guidelines
for quality assurance in cervical screening, 2nd draft of 15 December
2003.
12. Licker M, Moldovan R, Dragomirescu D, Brehar Cioflec D. Curs demicrobiologie specială, vol. II, Ed. Eurostampa, 2008 -15.
13. Mandell GL, Douglas RG, Bennett GE. Principles and Practice of Infectious Diseases, 7th Edition, Churchill Livingstone, 2009: 2035-49.
14. US Preventive Services Task Force. Screening for cervical cancer.
Recommendations and rationale. AHRG Pub. 03-515A, Jan 2003.
15. World Health Organization (WHO). Comprehensive Cervical Cancer
Control. A guide to essential practice. Geneva: WHO 2006.

PREVALENTA GENOTIPURILOR CU RISC CRESCUT A VIRUSULUIPAPILLOMA UMAN LA FEMEILE DE VARSTA FERTILA, IN PARTEA DE
VEST A ROMANIEIREZUMAT
Papillomavirusurile umane (HPV) sunt larg răspândite în populaţie, producând tumori epiteliale şi ale mucoaselor, şi sunt implicate
în patogenia afecţiunilor maligne genitale. Se cunosc mai mult de 100 de genotipuri HPV, din care aproximativ 40% infectează
mucoasa genitală.
În perioada 01.03.2007 – 19.09.2009, în cadrul laboratorului Bioclinica au fost testate 9245 de femei pentru detectarea infecţiei HPV.
38,27% dintre acestea au fost pozitive pentru cel puţin un genotip, dintre care 66,31% au fost genotipuri cu risc crescut. Extracţia
ADN-ului s-a efectuat în sistem automat (Magna Pure Roche), iar genotiparea cu LINEAR ARRAY HPV Genotyping Test (Roche).
Dintre genotipurile cu risc crescut, tipul 16 a fost detectat în cel mai mare procent (20,68%), urmat de genotipurile 31 (13,89%), 51(12,39%), 33 (8,75%), 66 (7,96%), 18 (6,78%), 52 (5,87%), 58 (5,37%), 68 (3,70%), 45 (3,55%), 39 (3,08%), 56 (3,05%), 59 (2,85%)
şi 35 (2,06%).
Cuvinte cheie: HPV, genotip, prevalenta, risc crescut

2011.21.2 (70)  Fiziologia - Physiology

35

PSYCHOLOGICAL CHARACTERISTICS AND PERSONALITYTRAITS IN PRODROMAL STAGES OF BODY DYSMORPHICDISORDER
DIANA JIVĂNESCU1, IULIA CRIŞAN2, MIRCEA LĂZĂRESCU1
1
Psychiatry Department, University of Medicine and Pharmacy Victor Babes Timisoara, Romania
2
Gataia Psychiatric Hospital, Romania

ABSTRACT
Objective: In a preliminary study on Romanian population, the authors examined the prevalence and correlates of certain psychological characteristics and personality traits regarding the Adonis Complex, a debilitating and chronic condition characterized by one’s
preoccupation with thoughts concerning muscle size and shape, as well as the specific behaviour that derives from this. Method:
Levels of anxiety and depression were investigated, as well as the prevalence of certain personality traits, similar to personality
disorders as described in the DSM-IV-TR and ICD-10, and several characteristics concerning the Adonis Complex. This study usedthe Hamilton Anxiety Scale, Beck Depression Inventory, the Altered Personality Questionnaire, and the Adonis Complex Questionnaire to characterize the diagnostic status of a population-based sample of 30 males who work out in fitness gyms at least 3 times
a week. Results: The presence of possible symptoms for muscle dysmorphia was significantly associated with the presence of mild
depression and moderate or severe anxiety symptoms. Significant correlations were found between depression and anxiety symptoms
and certain characteristics related to muscle dysmorphia. Research subjects also exhibited certain personality features, similar to themanic, emotionally unstable, paranoid and elated personality disorders. Conclusions: The authors found that the presence of muscle
dysmorphia characteristics was linked to the presence of depression and anxiety symptoms, which is similar to findings in other clinicalstudies. Their estimate of the personality trait prevalence is consistent with data from other studies and suggests a prevalence similar
to that of other serious psychiatric disorders (e.g., muscle dysmorphia comorbidity with personality disorders). These prevalence data
encourage further research in order to better understand and identify several characteristics of the Adonis Complex.
Key words: Adonis Complex, muscle dysmorphia, anxiety, depression, altered personality traits, avoidance behavior“While the general population is concerned with appearance, we are obsessed with it... I plan my meals.
I time them. I don’t eat for the pleasure of it. I eat according to my current goal“ (1)INTRODUCTION
The Adonis Complex, also known as reverse anorexia nervosa, bigorexia or muscle dysmorphia, can be defined as one’s
preoccupation with thoughts concerning appearance, especiallymusculature, as well as gaining weight without actually getting
fatter (2). According to Olivardia (3), diagnostic criteria for muscle
dysmorphia have three main components:
A. Excessive pathological preoccupation with thoughts
concerning muscularity, especially the idea that the body is notmuscular or lean enough;B. A clinically significant impairment in life activities (e.g.
decreased social functioning) as a result of the preoccupation
with insufficient musculature, namely:
1. Giving up on social/professional/enjoyable activities infavour of the compulsive need for strict weight-lifting schedule,
and a particular diet;
2. Avoidance of social situations that imply body exposure,and if such a situation is unavoidable, the individual experiences
intense subjective distress;
3. This preoccupation affects the individual’s social/professional/interpersonal functioning;
4. Continued harmful behaviours (e.g. excessive physicaltraining or strict diet) even if the individual is knowledgeable
about potential dangers, even if adverse reactions occur or he/she suffers physical damage.
A positive diagnosis of muscle dysmorphia requires thepresence of at least two out of four criteria.
5. Muscle dysmorphia and its associated behaviours are
based on the individual’s preoccupation with thoughts of not being muscular enough, and thus it can be differentiated from body
dysmorphic disorder or other eating disorders, if the associatedbehaviours are manifestations of a significant preoccupation
with body build or musculature as opposed to a preoccupation
with food intake.
According to Olivardia (3), muscle dysmorphia was primarily
diagnosed in 1997, in a historical context that witnessed a particular
growth in the amount of men concerned with their physical appearance, especially with their body size and form, particularly during
the last three decades of the 20th century. Thus, according to general statistics (4), during the last 30 years people’s dissatisfaction
with physical appearance has become three times more important(from 15% in 1972 to 34% in 1985, and to 43% in 1997).
Regarding the causes of this disorder, it is generally considered that there are a few possible risk factors which contribute

Received May 10th, 2011. Accepted June 1st, 2011. Address for correspondence: Diana Jivanescu, MD, Psychiatry Department, “Victor Babes” University of Medicine andPharmacy Timisoara, Eftimie Murgu Square No. 2A, Timisoara 300041, Romania; e-mail: dianajivanescu@yahoo.com

36

Fiziologia - Physiology  2011.21.2 (70)

to this disorder:
1. The genetic (biological) factor implies a certain serotonin irregularity, similar to that found responsible for obsessivecompulsive disorder (3,4);
2. Certain personal experiences in early childhood,mostly puberty and adolescence (bullying, teasing); lack of love
as a disharmony element responsible for the disorder, which maylead to rejection and low self esteem;
3. The socio-cultural factor, especially certain influences
through mass-media, promoting the so-called “real man”, who is
physically attractive, strong, with an extremely muscular body; ata very young age children come to interact with this over-sized
masculine figure, through several toys which represent certain
film/cartoon/story/comic book characters. In the 2000 book, The
Adonis Complex, Olivardia and Pope argue that muscle dysmorphia is fuelled by the portrayal of overly fit characters ofunattainable musculature in children’s cartoons, by observing
the changes their appearance has undergone since the ’70s,
from a so-called normal musculature to a hypertrophied body.
Another feature taken into consideration by these authors refers
to the fact that, during the past 30 years, women have gradually become more independent, witnessing a change in theirsocial status. Nowadays, not only men get to play the head of
the family part, by providing financial safety and social stability,
as most of the professional activities can be carried out by both
men and women. Thus, as the authors note, in search for their
manhood, men have developed a compensation mechanism, bybecoming more and more concerned with their bodies, especiallywith strength and bodily size; these features remain a malecharacteristic, due to the fact that “musculature is regarded asa symbol of manhood/masculinity” (4).
Several hypotheses regarding the origins of muscle
dysmorphia
The cognitive explanation is one of the most common
theories. It searches for possible causes of the disorder in a
cognitive vicious circle of dysfunctional thoughts. When a person
has a negative appraisal of internal body image, it influences theexternal representation of appearance. That triggers processing
self as an aesthetic object which results in negative internal body
image. So, people are not only sensitive to bodily cues, but also
threatened by them. In people vulnerable to somatoform disorders it can imply an over interpretation of bodily imperfections.
The psychodynamic explanation states that these areunresolved conflicts from childhood and extremely difficult feelings that are responsible for the disorder. The disorder provides ameans for people to express their emotions that otherwise would
be too difficult to express. In this case emotions are converted
into more tolerable physical symptoms. The purpose of such
conversion is to communicate extreme feelings in “physicallanguage”. Therefore a preoccupation with musculature could
be treated as an individual’s unconscious displacement ofsexual or emotional conflict (or feelings of guilt, or even poor
self-image).
The cognitive-behavioural explanation states that the2011.21.2 (70)  Fiziologia - Physiology

responsibility for muscle dysmorphia is shared by: cultural
factors, biological predispositions, psychological vulnerabilities
and early childhood experiences. Cultural factors manifest themselves in an exaggerated emphasis on appearance, physicalstrength and attractiveness. People compare themselves withothers and even little deviations from the pattern perceived as
ideal lead to extremely negative appraisals. Next, some people
are biologically predisposed to a constant drive for perfection.
Psychological vulnerability is mostly based on low self-esteem.
Childhood experiences connected with the disorder are: disharmonious family background, bullying, teasing. This may produce
feelings of being unloved, insecure and rejected.
According to the biological explanation a serotonin irregularity is mostly responsible for the disorder.
The evolutionary perspective links the causes of thisdisorder to the progressive alignment of men and women in most
aspects of every day life. Due to women’s social emancipation,
men have gradually lost their traditional identity as food andincome providers, family protectors and potential fighters. While
women gained in independence, men began to suffer a lack ofidentity, and secondarily physical appearance became more andmore important to them. The need for redefining masculinity canbe seen as the need for increasing muscularity, as the latter isconsidered to be a symbol of masculinity.
Regarding the prevalence of this disorder, muscle dysmorphia is most common in males and often starts in the lateteens, although sometimes, they can be older. Olivardia andothers found in a 2000 study on American populations that the
average onset age was 19.4 years (3). The preoccupation withmuscularity is considered more frequent in young males, incomparison with men over 45 years of age who show a lower
degree of concern. However, the desire to work out the upper
body seems to appear in younger boys as well (between 5 or
6 years of age) (4).
From a psychopathological point of view, muscle dysmorphia involves several changes on the following levels of
psychological functioning:
A. THE COGNITIVE LEVEL – psychopathology changes
regarding the following psychological processes:
1. Perception – pathological delusions regarding one’s
body (the individuals perceive themselves as too skinny andunderweight);
2. Attention – selective attention, prevailing long-term focus
on one’s body (individuals selectively focus their attention on
perceived physical “defects”, they are hypervigilant to even small
deviations from perceived ideal and they ignore information that
their body image is not consistent with reality);
3. Cognition – pathological changes such as:a. Delusional thoughts and beliefs (negative, distorted andrepeated convictions regarding one’s physical appearance, witha tendency towards certainty and incorrigibility);b. Recurrent thoughts of worthlessness, helplessness,
hopelessness, and dissatisfaction;c. Self criticism;d. Obsessive thoughts concerning muscular growth, the37need for excessive exercising, and a special high-protein diet;e. Low self-esteem;
f. Frequent comparisons with others or with certain so-called“ideal” figures.
B. THE EMOTIONAL LEVEL – individuals frequently exhibit
a low mood, irritability, and melancholy. Also, they often experience intense feelings of dissatisfaction related to their perceived
appearance, self-hatred, as well as strong feelings of shame.
Other common symptoms include anxiety and subjective distress, as well as chronic feelings of emptiness. In severe cases,
individuals may exhibit symptoms of phobia, characterized byintense fear of not living up to other people’s expectations.
C. THE BEHAVIORAL LEVEL – refers to regular andexcessive physical training, as well as keeping a strict diet.
Nowadays it is generally considered that muscle dysmorphia
is based on a negative body image which derives from:•the excessive, time consuming preoccupation withbodily size and form, causing significant social and professional
impairment;•
dissatisfaction with one’s perceived body image, causing low self esteem, depression, and social anxiety.
In this regard, several studies assert the fact that one’s
biased perception of the actual size and shape of their own
body may be based on certain cortical deficits associated withcertain emotional processes that enhance one’s tendency tofocus exclusively on the size and shape of their bodies. Thus,
specific perceptive disturbances related to one’s body image
should ultimately be seen as coming from certain cortical
deficits which in turn cause visual and spatial disturbances; allin all this perspective might provide an answer to why certain
people tend to either underestimate or overestimate their bodily
features (5).
In term of clinical presentation, symptoms vary in severity
on a continuum. Common symptoms are:
• Frequent and repeated mirror and other reflective surfaces
checking;• Periodic measurements of the shape and muscle mass;•
Preference for clothing designed to hide the physicalform (large clothes);• Avoiding situations requiring body exposure;• Concern to engage regularly and rigorously intense exercise/ excessive (daily, several times a day);• Physical training in spite of physical injury ( muscle tear,stretching of ligaments, tendons or joints);• Ignoring/ avoiding social or family events and job responsibilities in order to exercise;• Main concern for a strict diet rich in protein and fat-freeand sticking to this;• Constant dissatisfaction related to the shape and size ofmuscle mass;• Consumption of anabolic steroids (amphetamines, HCG,
L-DOPA, estrogen blockers, steroids).
The benefits of this behaviour (efficiency in building muscle
mass, muscle sustainability) are accompanied by a series of side
effects, among which we mention:
38• Non-psychiatric effects: increased risk for cardiovasculardisease, stroke, prostate cancer;• Psychiatric effects: irritability, emotional instability, tendency to aggressive manifestation, verbal and physical aggression,
impaired reasoning, megalomaniacal ideation, severe depressive
episodes, physical and psychological dependence.
In severe cases, the concern for physical exercises (training)
becomes an obsessive idea and the urge to perform theme turns
in to compulsion. Missing a training session causes intra-psychic
tension and anxiety, accompanied by feelings of vulnerability,
ideas of incapacity and guilt.
What comorbidity is concerned, muscle dysmorphia ismost frequently associated with mood disorders (depression or
bipolar disorder), anxiety disorders, eating disorders, obsessivecompulsive disorder, schizophrenia, Cluster B personality
disorders (particularly Narcissistic personality disorder type)(6). Approximately 58% of patients with muscle dysmorphia
have been found to suffer from mood disorder like unipolar
depressive disorder or bipolar disorder (hypomanic episodes
while using steroids) (3).
There is still a debate about comorbidity with anxiety disorder or classification in anxiety disorder group. Thus, patient
suffering from bigorexia show anxiety symptoms, especiallywhen they are unable to meet rigorous physical training program and diet. According to the literature, anxiety disorder arepresent in 29% of those with muscular dysmorphia (3), while
statistics show that the same percentage of those diagnosed
with Adonis Complex describe a history of anorexia or bulimia
nervosa (3). Despite these data, the two disorders differ in thatthe characteristic manifestation of generalized anxiety disorder(muscle tension, irritability, psychomotor restlessness, sleep
disturbances, etc.) are not necessarily manifest in those withmuscle dysmorphia.
There are also certain similarities between muscle dysmorphia and obsessive-compulsive disorder, regarding certain
clinical features, namely: obsessive thoughts (related to muscular
growth or not being muscular enough), compulsive behaviour(such as excessive physical training, adherence to a strict diet),feelings of discomfort, anxiety, distress, low mood particularly
when individuals find themselves incapable of following theirstrict training or dieting schedule.
Some studies have found certain similarities between
muscle dysmorphia and social phobia, regarding individuals’
reaction in certain social situations that require dealing with otherpeople’s attention (7).
In addition to this, some authors have found several similarities
as well as differences between muscle dysmorphia and cluster B
personality disorders (particularly narcissistic personality disorder)(7). On the one hand, both categories of disorders refer to identity
related concerns as well as unstable interpersonal relations; on theother hand, while narcissistic individuals have a need for being thecentre of attention and for being socially successful, individuals
with muscle dysmorphia avoid similar situations consciously andknowingly, for fear of rejection, thinking that others might noticeand mock their lack of muscularity. Also, individuals with muscle
Fiziologia - Physiology  2011.21.2 (70)dysmorphia often report working out in order to gain acceptancefrom others rather than exhibiting ideas about their influence onothers, as in the case of the narcissistic psychopath. Finally, individuals with muscle dysmorphia don’t usually exhibit the impulsive
behaviours common to cluster B personality disorders.
There are also several features which link muscle dysmorphia to eating disorders, such as: disturbed perception of bodily
size and shape (8); feelings of lack of control related both to binge
eating periods (in bulimia nervosa) and to the incoercible needfor training and dieting (in muscle dysmorphia); similar aetiology which includes: dissatisfaction with one’s body image, lowself esteem, emphasis on social and cultural influence (mainly
through mass-media). However, on a behavioural level, these
disorder types involve distinctive compensatory behaviours,
namely: fasting (abstinence from food) in anorexia nervosa,eating according to a special high-protein and low-fat diet inmuscle dysmorphia, and the use of laxatives, enemas or diuretics
to avoid gaining weight in bulimia nervosa.
Last but not least, individuals with muscle dysmorphia frequently end up neglecting work, school, family, and other social
aspects in life because of excessive exercising in order to continuously gain muscle size/strength; this type of secondary behaviour
turns out to be repetitive (1 to 5 training sessions each day), as wellas time consuming (more than 200 minutes each day) (4).
Due to the fact that muscle dysmorphia was only recently
described and diagnosed as a mental disorder, there has been
considerable controversy over the effectiveness of certain treatment methods. Nowadays, the complete treatment regimen formuscle dysmorphia requires a multidisciplinary approach, involving a team of mental health professionals, including psychiatrists,
nutritionists, and psychologists (9). Patients are usually required
to fill out several questionnaires in order to assess both individual
characteristics and different levels of severity before a specifictreatment is instituted.
MATERIALS AND METHODS
The sample involved in the present study consisted of 60
male subjects between 18 and 40 years old, who were divided
into two samples, as follows: the research sample consisted of
30 subjects who attend body-building gyms at least 3 times aweek, for at least 1 year, and who spend at least 1 hour a day
performing physical training in order to improve their physicalappearance (not for taking part in competitions). The objectiveof the present study was investigating whether there would
be any cases of muscle dysmorphia (mostly in early stages ofdevelopment) among these men. The second sample, the socalled control sample, consisted also of 30 individuals who wereselected following certain sample criteria, such as: gender, age,marital, and professional status.
Both samples had to fill out the following standard questionnaires: the Hamilton Anxiety Rating Scale, the Beck Depression
Inventory, a screening questionnaire regarding possible issues
related to physical appearance, the Adonis Complex Questionnaire, and the Altered Personality Questionnaire (H. Schmies2011.21.2 (70)  Fiziologia - Physiology

check). Regardless what sample they belonged to, all subjects
had to fill out each of the questionnaires.
RESULTS
1. Comparative sample analysis. Socio-demographic
features
The mean age of the research sample was 22.9 years (witha standard deviation of 3.67), while for the control sample themean age was 23.96 years (with a standard deviation of 13.64).
Comparative sample distribution is shown below:Age<21 years old
21-30 years old
31- 40 years old

Research sample
Subjects. no %
3
10.00
26
86.67
1
3.33

Control sample
Subjects. no %
3
10.00
26
86.67
1
3.33

According to stratified sampling, the percent distribution ofage categories is the same in both the control and the researchsample.
Due to the fact that both the population mean and thestandard deviations are unknown variables, associated with theclinical nature of the research sample, and the construction of
the control sample using quote sampling, the Liliefors test had
to be used in order to estimate data variance. As a result thenormal distribution hypothesis proves false, thus requesting theuse of non-parametric tests for data analysis.
Clinical parameters analysis
Levels of anxiety, as measured with Hamilton Anxiety
Scale
The Hamilton Anxiety Scale consists of 14 items that quantify several anxiety related features, such as: anxious mood, fear,
insomnia, cognitive symptoms, depression, various physicalbehaviours and symptoms, and muscular strain. Each item isscored on a scale of 0-5 (0 = lack of symptoms, 5 – severe andimpairing symptoms). The scores range from 0 to 56, any scorehigher than 14 indicates clinically significant anxiety, dividing
the remaining categories as follows: 14-17 for mild anxiety
levels, 18-24 for moderate anxiety levels, and 25-30 for severe
anxiety levels.
a) Case distribution in the research sample depending on
anxiety levels is shown below:
Research sample
Subject Cumulative Percent
no.
frequency
Minimal or no 25
25
83.3
anxiety (score <14)
Mild anxiety level 1
26
3.3(scores 14-17)
Moderate anxi- 3
29
10.0
ety level (scores
18-24)
Severe anxiety 130
3.4levels (scores 2530)
Anxiety level

Cumulative
percent
83.3
86.6
96.6
100.0

39

Up to this point research data show that 13.4 % of thesubjects included in the research sample exhibit moderate andsevere levels of anxiety.
b) Case distribution in the control sample depending on
anxiety levels is shown below
Anxietylevel
Minimal orno anxiety(score < 14)
Mild anxiety
level (scores14-17)
Moderateanxiety level(scores 1824)
Severeanxiety levels(scores 2530)

Control sample
Subjects
no.

Cumulative
frequency

Subjects
no.

29

29

96.7

Cumulative
percent
96.7

1

30

3.3

100.0

0

30

0.0

100.0

0

30

0.0

100.0

None of the subjects in the control group exhibits severeor even moderate anxiety, while only one subject was found toreport mild anxiety; all in all, 96.7% of subjects in the control
sample exhibit no clinically significant anxiety. The difference
between the two percentage values (13.4% vs. 0.0%) is statistically significant (p = 0.042), thus results indicate that researchsubjects exhibit moderate and severe anxiety in a significantly
greater respect than control subjects.
By comparing the mean values between the two samples,
we get the following results which are shown below:
SampleMedianValue
Control sample 3.0
Research sample 2.0

Minimal
value
0.0
0.0

Maximum
value
14.0
28.0

Sum of ranks
in the researchsample929.5

UZ

p-level435.5-0.214p=0.826>0.05
not statisticallysignificant

Sample differences are not statistically significant, showingthat all in all there are no notable differences between the two
samples concerning anxiety levels. Yet we believe that this result
can be determined by the relatively small number of subjects
included in the present study.
Levels of depression, as measured with Beck Depression
40

The inventory contains 21 questions, each answer beingscored on a scale value of 0 to 3. The cut-offs used differ fromthe original: 0–13: minimal depression; 14–19: mild depression;
20–28: moderate depression; and 29–63: severe depression.
Higher total scores indicate more severe depressive symptoms.
Level categories are as follows:
0.5 – 1.2 – Mild depression
1.2 – 2 – Moderate depression
2 – 2.5 – Severe depression
2.5 – 3 – Severe depression, risk of suicide
The sample situation is as follows:
SampleControl sample
Research sample

Median
value
0.28
1.0

Minimum

Maximum

0.0
0.0

1.67
2.19

Standard
deviation
0.39
0.65

Results show the fact that the mean value in the researchsample as well as the control sample indicates the absence ofdepression (<0.5).
A chart showing these differences is given below:

Standard
deviation
4.36
8.10

Thus, in spite of the small number of subjects in both samples, subjects in the research sample tend to display moderateand severe anxiety in a greater proportion than the control
sample, keeping in mind that this is not the case of a normal
distribution of variables. We believe that by including more casesin the analysis we could get more significant results, concerning
differences between the two samples.
Results of the non-parametric Mann-Whitney U test are
as follows:
Sum of ranks
in the control
sample900.5

Inventory

To determine whether the above mentioned differences
are of statistic significance, we used the non-parametric MannWhitney U test. Results are shown below:
Sum of ranks
in the control
sample1118.0Sum of ranks
in the researchsample712.0

UZ

p-level

247.0

3.00

p=0.002<0.05
Statisticallysignificant

There is a statistically significant difference concerning depression between the two samples, namely general depression
level is higher in the research sample than in the control sample,
corresponding to a mild depression level (0.92).
Comparing frequencies of altered personality traits in thetwo samples
Considering the particular way of scoring and interpreting
the Altered Personality Questionnaire, we compared frequencies
in the two samples, concerning the prevalence of all 10 personality types and their corresponding features.
Fiziologia - Physiology  2011.21.2 (70)Generally a particular personality feature is taken into consideration if subjects report at least 50% of its characteristics (oscore of 50% or more). Results are shown below:
Personality feature

I – Histrionic
II – Obsessive-compulsive
III – Paranoid
IV – Borderline
V – Manic
VI – Low mood
VII – Emotionally unstable
VIII – Elated
IX – Anxious
X - Emotional

Research
sampleSubjects %
no.
17
56.7
12
40.0
22
73.3
14
46.7
30
100.0
10
33.3
22
73.3
26
86.7
10
33.3
21
70.0

Control sample
Subjects
no.
18
11
22
12
24
7
16
28
13

28

%

elated, and paranoid personality type are also present in thecontrol sample (60% of the subjects); however, taking the following graph into account, all altered personality traits (except
those corresponding to the elated type) are less frequent in thecontrol sample than in the research sample. The χ2 test does notshow significant differences between frequencies.

60.0
36.7
73.3
40.0
80.0
23.3
53.3
93.3
43.3
93.3

A graph showing personality profiles for both samples isshown below:

Thus, in the research sample there is a prevalence of
personality traits corresponding to following personality types:
manic (100%), elated (86.7%), paranoid (73.3%) and emotionally unstable (73.3%). These are common features in 60% of
the research subjects.

Several personality features, corresponding to the manic,
2011.21.2 (70)  Fiziologia - Physiology

Thus, results indicate that personality features related to themanic, emotionally unstable, low mood, obsessive-compulsive,and borderline personality types prove to be more frequentin the research sample, while in the control sample there is ahigher frequency regarding other personality types, such as theemotional, anxious, elated, and histrionic type.
All in all, most important frequency differences were foundbetween the two samples, namely concerning the manic personality type (100% in the research sample vs. 80% in the control
sample), as well as the emotionally unstable type (73.3% in the
research sample vs. 53.3% in the control sample). According to
the classical interpretation of this questionnaire, we have identified several altered personality traits in the research sample, corresponding to the manic, paranoid, elated, emotional, emotionallyunstable, and histrionic types, while results regarding the control
sample indicate other features, related to the emotional, manic,paranoid, and emotionally unstable personality types.

As far as the research sample is concerned, one cannotice the high intensity of certain personality features, likethose corresponding to the manic, paranoid, and emotionally41

unstable types; the comparison test Mann-Whitney indicatesstatistically significant differences between the two samples
only what the manic personality type is concerned. Results areshown below:
Altered personal- Sum of
ity featuresranks
in theresearch
sampleI – Histrionic
II – Obsessivecompulsive
III – Paranoid
IV – Borderline
V – Manic

VI – Low mood
VII – Emotionallyunstable
VIII – Elated
IX – Anxious
X - Emotional

Z

920.0

Sum of U
ranks
in thecontrol
sample910.0 445.0

p

933.0

897.0

432.0

0.266 0.790

993.0
1001.0

837.0
829.0

372.0
364.0

1121.0

709.0

244.0

905.5

924.5

440.5

1.153 0.249
1.276 0.203
0.002
Statisti3.047
callysignificant-0.140 0.883

982.5

847.5

382.5

0.998 0.318

969.5
858.5
803.5

860.5
971.5
106.5

395.5
393.5
338.5

0.806 0.424-0.835 0.403-1.648 0.099

0.074 0.941

All in all, we can conclude that subjects in the researchsample (Adonis complex) exhibit certain personality traits corresponding to the manic type more frequently and at a higher
intensity level than subjects in the control sample.
The Adonis Complex Questionnaire
The questionnaire contains 13 items, each with 3 possible
answers, scoring from 1 to 3 (answer a = 1, answer b = 2,answer = 3), corresponding to the frequency/intensity of eachmeasured factor.
The measured features are as follows:• subject’s preoccupation regarding physical appearance(amount of time spent thinking about this issue), as well as the
feelings involved;• active avoidance behaviour due to this preoccupation;• active behaviour in order to correct the perceived defect(amount of time spent exercising, dieting, consumption of dietsupplements);• social impairment due to subject’s behaviour and beliefs
related to muscle size and shape (amount of money spent,
avoidance of interpersonal intimacy, social impairment);• risk behaviour: health issues related to the above
mentioned behaviour (substance abuse, extreme training
methods).
The total score is obtained by adding the scores obtainedfor each of the 13 items.
What our sample is concerned, results show that there are
more subjects in the research sample than in the control sample
who: spend more time worrying about their physical appearance,
more frequently feel unhappy due to this preoccupation; spend
42

more time performing grooming activities and physical training,and more frequently tend to abuse dietary supplements in orderto improve muscle size and shape.
These preoccupations related to physical appearance
greatly influence subjects’ daily life: most of the subjects in the
research sample (13.3% vs. 0% in the control sample) report thatthe amount of money spent in order to improve their appearance
have frequently caused financial issues. In addition to this, mostsubjects in the research sample have report that this preoccupation has „sometimes” or even „frequently” affected their social
interactions, the level of intimacy, and work performance, as well
as several other social activities.
What risk substance consumption is concerned, both
samples reported „never/rarely” or „sometimes” exhibiting thisbehaviour, with comparable scores in both cases.
All in all, results indicate a distinct profile for subjects in
the research sample, compared to those in the control sample,
featuring: excessive preoccupation for muscle size and shape,social impairment, active behaviour used for mending this issue, namely a special diet, as well as intense and excessive
physical training.
In order to check if there is a significant difference between
the two samples, we used the Mann-Whitney comparison test.
Results indicate significant differences regarding the intensityof preoccupations related to physical appearance, prolonged
physical training, as well as the total score.
Correlations between general test scores obtained by
the research sample (subjects with muscle dysmorphia)
We used the correlation test Spearman R in order to check
whether there are any significant correlations between subjects’scores regarding the evaluation methods we used. Results
indicate the following significant correlations:• between the general BDI score and the total Hamilton
score (R = 0.599, p <.05), namely subjects who exhibit higher levels of depression also tend to report higher levels of anxiety;• the more severe a subject’s level of depression, theweaker the belief that his physical appearance is normal (R =-0.644, p <.05);• the more severe a subject’s anxiety level, the more he
will try to avoid social contact (R = 0.414, p < 0.05), make useof more methods in order to improve muscle size and shape (R= 0.375, p < .0.05), feel more unhappy because of excessive
preoccupation related to physical appearance (R = 0.421, p <.05), and avoid being seen by others (R = 0.431, p < .0. 05).
DISCUSSION
Following data analysis up to now, several conclusions canbe drawn, regarding the existence and possibility of identifying
certain characteristics, as well as personality traits concerning
individuals who might suffer from the Adonis Complex.
Regarding the two samples involved in this study, results
indicated the following:
1. Subjects in the research sample exhibit higher levels ofmoderate and severe anxiety than subjects in the control sample.
Fiziologia - Physiology  2011.21.2 (70)

2. General depression level in the research sample proves
higher than in the control sample (mild depression vs. absenceof depression).
3. 60% of the subjects in the research sample exhibit certain
altered personality features, mostly corresponding to the manic,elated, paranoid and emotionally unstable types; these features,
excluding those related to the elated type, prove to be less frequent in the control sample. Significant differences were foundbetween samples only related to the manic personality type, withhigher scores in the research sample.
4. In comparison to the control sample, subjects in the research sample also tend to: spend more time worrying about theirappearance, feel unhappier because of their excessive preoccupations with muscle size and shape, spend more time exercising, andare more prone to specific dietary supplement consumption.
5. In addition to this, subjects with muscle dysmorphia reporthigher levels of anxiety and depression, correlated with a negative
self evaluation pattern, as well as a greater level of distress causedby excessive preoccupation related to their physical appearance,sustained by a tendency to avoidance behaviour.
As shown above, results are consistent with other researchfindings on muscle dysmorphia, even if our samples lack aconsistent number of subjects, and our study variables indicate
the prevalence of certain aspects only in a small degree. In thiscase this preliminary study allows us to extend our researchand investigation, by adding a larger number of participants,
more complex evaluation measures, as well as by sample

diversification. Future research will be conducted according tothese findings.
REFERENCES

1. McBride H. Obsessed with size: ‚Bigorexia’ sufferers develop
unhealthy compulsion to exercise, 2006, found at http://www.eatingdisorder.com/Eating-Treatment/Eating-Disorders/muscle-dysmorphia- bigorexia.htm.
2. Morgan JF. From Charles Atlas to Adonis complex – fat is more thana feminist issue. The Lancet, 2000; 356 (9239): 1372-73.
3. Olivardia R. Mirror, mirror on the wall, who’s thelargest of them all?
The features and phenomenology of muscle dysmorphia. Harvard
Review of Psychiatry, 2000; 9:254-9.
4. Pope HG Jr, Phillips KA, Olivardia R. The Adonis complex: The secretcrisis of male body obsession. New York: The Free Press, 2000.
5. Thompson JK, Spana RE. The adjustable light beam method for theassessment of size estimation accuracy: description, pscyhometrics,
and normative data. International Journal of Eating Disorders., 1988;
7: 521-526.
6. Phillips KA. The Broken Mirror. New York: Oxford University Press,
1996.
7. Perez-Lopez MS, Petretic P. The etiology and maintenance of body
imagedissatisfaction in men and women: A review of the literature. In
S. P. Shohov (Ed.). Advances in Psychology Research (Volume 28, pp.
57-102). Huntington, N. Y.: Nova Science Publisher, 2004.
8. Cash TF, Pruzinsky T. Body Image. The Guilford Press, New York
London, 2002.
9. Dawes J, Mankin T. Muscle dysmorphia. Strength and Conditioning
Journal, 2004: 26: 24-25.

CARACTERISTICI PSIHOLOGICE ŞI TRĂSĂTURI DE PERSONALITATE
ÎN COMPLEXUL ADONISREZUMAT
Obiective: În acest studiu preliminar pe populaţie românească, autorii au investigat prevalenţa şi corelaţiile între anumite aspecte
psihologice şi trăsături de personalitate implicate în circumscrierea diagnostică şi clinică a Complexului Adonis (dismorfiei musculare). Metodă: Au fost analizate nivelele de anxietate şi depresie, prevalenţa anumitor trăsături accentuate de personalitate, similare
celor descrise la nivelul tulburărilor de personalitate din DSM-IV-TR şi ICD-10, precum şi frecvenţa anumitor caracteristici specificeComplexului Adonis la un lot de 30 de subiecţi de sex masculin. Studiul a utilizat Scala de Anxietate Hamilton, Inventarul de Depresie
Beck, Chestionarul pentru Personalităţi accentuate şi Chestionarul pentru evaluarea complexului Adonis, pentru a caracteriza lotul destudiu, alcătuit din 30 de bărbaţi care frecventează sălile de fitness de cel puţin 3 ori pe săptămână. Rezultate: Prezenţa posibilelor
simptome specifice complexului Adonis a fost asociată cu nivele uşoare respectiv moderate ale anxietăţii, respectiv cu simptomeuşoare ale depresiei. La nivelul lotului de studiu s-au înregistrat anumite trăsături accentuate de personalitate, corespondente
tulburărilor de personalitate hipertime, paranoide, ciclotime şi borderline. Concluzii: Rezultate studiului permit asocierea anumitor
caracteristici atribuite complexului Adonis cu indici de depresie şi anxietate la nivel subclinic. Aceste rezultate sunt similare cu altestudii. Prevalenţa trăsăturilor de personalitate se încadrează pe direcţia cercetărilor anterioare, sugerând comorbiditatea tulburării
cu anumite tulburări de personalitate. Aceste concluzii permit efectuarea de cercetări viitoare cu scopul de a institui noi metode mai
eficiente de a depista şi eventual interveni în cazul persoanelor ce suferă de Complexul Adonis.
Cuvinte cheie: Complexul Adonis, dismorfie musculară, anxietate, depresie, personalitate accentuată, comportament secundar

2011.21.2 (70)  Fiziologia - Physiology

43

PARTICULARITIES OF LIPID METABOLISM DISORDERS
IN PATIENTS WITH DIABETES MELLITUS AND THYROIDDISEASEADRIANA GHERBON, LAVINIA NOVEANU, GEORGETA MIHALAŞ
Department of Physiology, “Victor Babeş” University of Medicine and Pharmacy Timisoara

ABSTRACT

Increased serum cholesterol, triglycerides, or both, as well as low HDL cholesterol, characterize dyslipidemia. They may be primary(genetic) or secondary. Their diagnosis is usually biochemical by determining total cholesterol, triglycerides and various lipoprotein
fractions. Lipid metabolism disorder treatment consists in lifestyle changes, exercise and administration of lipid-lowering drugs. The mainconsequence of appropriate untreated dyslipidemia is appearance of cardiovascular disease or worsening of a preexisting one.
Secondary dyslipidemia represent the diseases frequently associated with diabetes mellitus and thyroid disorders. The purpose ofthis study was to determine the main types of lipid metabolism disorders seen in patients with different changes in glycemic balance
and thyroid disorders. General group studied was represented by 733 cases, aged 7-79 years. The study group was subdivided
by age criterion in two groups: children and adult group. They used clinical, radiological, biochemical, hormonal, immunologicalparameters.
Keywords: diabetes mellitus, thyroid diseases, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, cardiovascular disease

INTRODUCTION
Lipid metabolism disorders are frequent diseases in the general population. Increased serum cholesterol (TC), triglycerides(Tg) or both, as well as low HDL-cholesterol (HDLc) characterizes
the lipid metabolism disorders. They may be primary (genetic)or secondary. The secondary one is frequently associated with
unbalanced diabetes mellitus, obesity, sedentary lifestyle, being
more common in industrialized than in developing countries. In
all epidemiological and interventional studies, they represent arisk factor for coronary heart disease. Their diagnosis is usually
biochemical by determining total cholesterol, triglycerides andvarious lipoprotein fractions. Lipid metabolism disorder treatment consists in lifestyle changes, exercise and administrationof lipid-lowering drugs.
Initial the lipid metabolism disorders were classified byFredrickson on ultra centrifugation followed by electrophoresis
in five types: type I, IIa, IIb, III, IV and V.
Type I may be primary or secondary (very rare). It is due tofamily deficiency of LPL (lipoprotein lipase), of apoprotein C II
or of liver triglycerides lipase (HPL). The disease is rare, being
described, in total, about 100 cases to date, and is manifested
by episodes of abdominal pain (with an aspect of acute abdomen
when the Tg exceed 1500 mg %), recurrent, due to a marked
increase in chylomicrons.
Type IIa is primary in the most majority of cases (essential
hypercholesterolemia). Familial forms appear through mutations

Table I. WHO classification of hyperlipoproteinemias (HLP) (modified) (17)of the responsible gene of LDL receptor synthesis, whose totalor partially deficits determine remain of CT outside the cells,
resulting in the appearance at the offspring that inherit the gene,of the early and severe arteriosclerosis.
Type IIb is present in 0.5% of the population. The primaryfamily form is called familial combined HLP. Genetic defect is
not precisely known, it seems that this is an increased of hepatic
synthesis of apo B100. The third of those who inherit this tare, theclinical- biologically type is IIa, at the third IIb and at the rest type
IV. Even at the same person may be encounter different types at

Received March 11th, 2011. Accepted April 25th, 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

44

Fiziologia - Physiology  2011.21.2 (70)different times, depending on influence of environmental factors,
diet; characteristic for this type is early arteriosclerosis.
Type III (yellow palms disease, named due by palmarydigital xantomatosis) is rare (1 in 5000) and, in majority, primary.
Genetic defect is abnormal inheritance of apoE, which is notrecognized by specific hepatic receptors.
Type IV is quite frequently (0.2 to 0.3%) and can be primary(familial endogenous hypertriglyceridemia - unknown genetic defect)or secondary, carbohydrate-induced, were the most frequently
associated with diabetes mellitus, but also with obesity, food rich incarbohydrates, alcoholism and estrogen contraceptives.
Type V (endo-and exogenous familial hypertriglyceridemia)
is rare (1/106 subjects). Genetic defect is not precisely known,
and the symptoms are similar to type I. Atherosclerotic events
may exist, but are rare than in types II, III and IV.
The main causes of lipid metabolism disorders are
represented by diabetes mellitus, obesity, hypothyroidism andnephrotic syndrome. The consumption of drugs, alcohol, foodrich in carbohydrates, acute pancreatitis and pregnancy may
also determine it.
Unbalanced diabetes mellitus (DM), both type 1 and type 2,
is one of the most common causes of hypertriglyceridemia, which
may be more severe in the presence of ketosis. Usually after
restoration of glycemic balance by insulin (in type 1 diabetes) ororal drugs (OAD) (in type 2 diabetes) is restore also the normal
blood lipid values.
Hypothyroidism is frequently associated with increased LDLc,
mixed dyslipidemia and sometimes with isolated Tg increases.
Nephrotic syndrome is commonly associated with increased
LDLc, mixed dyslipidemia and sometimes with isolated Tg
increases. Increased levels of proteinuria are correlated withthe severity of hyperlipidemia.
Some medications such as high doses of thiazide diuretics
or clorthalidon, high doses of beta-adrenergic blockers,
except those with intrinsic sympathomimetic activity, estrogen
replacement therapy, and oral contraceptives containing highdoses of estrogen, tamoxifen and corticosteroids are involvedin occurrence of lipid metabolism disorders.
Other causes of lipid metabolism disorders are represented
by excessive alcohol consumption, diet rich in carbohydrates (>60% of calories), acute pancreatitis and pregnancy.
The prevalence of lipid metabolism disorders in U.S. isabout 10% in men aged over 30 years and in women aged over
55 years. (5)The prevalence of severe hypertriglyceridemia, characterizedby values of Tg over 2000 mg /dl, is estimated at 1.8/10 000 at
adult white race, their prevalence is higher in patients withdiabetes mellitus and alcoholism. (5)The lipid metabolism disorders are associated with anincreased risk of cardiovascular disease, especially in case of lower
HDL and/or increase LDLc. If these changes are controlled, somestudies have shown that elevated Tg values do not correlate withan increased risk of cardiovascular disease. (12). Other studies
suggest, however, that elevated Tg is an independent risk factor for
cardiovascular disease. (3). Since the metabolism of triglycerides
2011.21.2 (70)  Fiziologia - Physiology(chylomicrons, VLDL) and HDL metabolism are interdependentand because of Tg level lability, impact of hypertriglyceridemia
on cardiovascular risk is difficult to confirm. Randomized clinical
trials have used medication that lowers Tg showed decreased
coronary events in primary and secondary prevention programsfor coronary artery disease. (4, 6, 9, 12)
If the Tg value growth over 1000 mg/dl may appear acutepancreatitis.
Regarding race, it was found that lipid metabolism disorders
are more common in Africans than in white American race. (5)In the prospective Cardiovascular Munster study (PROCAM),
medium hypertriglyceridemia was more common in men (18.6%)
than in women (4.2%). (1)The lipid metabolism disorders are more common in males
until age 50 years, and then slightly decreases. In women, they
continue to increase with age. The Tg values over 150 mg/dl are
more common in men from age 30 years and in women fromthe age of 60 years. (5)MATERIAL AND METHODS
Investigated population
The study included subjects with diabetes mellitus,
which in time present thyroid disease, or subjects with thyroid
disease who subsequently present diabetes mellitus.
The study group comprised 733 cases aged 7-79 years.
Subjects were divided as follows:• group of children that included 83 children and adolescents
aged 7-17 years (14.57 ± 2.25 years), with a ratio F/M of
5.9/1.
• group of adults that included 650 adults aged 18-79 years(52.03 ± 12.46 years), with a ratio F/M 9.48/1.
Methods of investigation
Methods of investigation were the clinical data - history,
present status, and imaging - ultrasound thyroid, biochemistry- carbohydrate metabolism parameters: fasting blood glucose,urine glucose, glycosylated hemoglobin and thyroid hormone
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 mellitus fasting blood glucose values above 126 mg%, impaired glucose
tolerance - fasting glucose values between 110-126 mg% and theoral glucose tolerance test (OGTT) at 2 h between 140-200 mg%
and fasting impaired glucose tolerance - fasting glucose values
between 110-126 mg% and OGTT at 2 h under 140 mg%.
Determination of glycated hemoglobin (HbA1c) was
achieved through the DiaStat program for glycated hemoglobin
HbA1c who measures the ratio of glycated hemoglobin to total
HbA.
Determination of serum levels of TSH, free fraction ofserum level of triiodothyronine (FT3), free fraction of thyroxin(FT4) were ARCHITECT quantitative method, which is an
immunologic determination by chemiluminescence’s with small
45

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.
Immunological parameters were represented by somemarkers of thyroid autoimmunity – antiTPO and antiTG antibodies(AB). To determine the serum titers of AB antiTPO Axsym
antiTPO kit was used, the method is enzyme immunoassay withmicro particles, Meia (Micro particle Enzyme Immunoassay). It
was considered normal: AB antiTPO (<35 IU/ml). To determine
the serum titers of AB antiTG Axsym antiTG kit was used, themethod is enzyme immunoassay with micro particles, Meia
(Micro particle Enzyme Immunoassay). It was considered
normal: AB. antiTG (<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 withcervical anatomical structures and thyroid parenchyma changes.
Appearance of normal thyroid parenchyma is characterized by ahigh intensity echogenic, homogeneous, easily distinguishable
from the neck muscles, which look hypoechogenic. Inflammatory
and autoimmune processes are hypoechogenic. The degree ofthyroid hypoechogenity was assessed as: discreet +, moderate+ + and marked + + +. In autoimmune thyroid disease is foundhypoecogenity of thyroid parenchyma. The Graves’ disease
appears: thyroid volume generally increased, hypoechogenity
with different intensities with variable homogeneity. Chronic
autoimmune thyroiditis appears: hypoechogenity uneven andnormal or increased thyroid volume.
The “fasting” lipid profile in peripheral blood was assessedby determining the total cholesterol (CT), triglycerides (Tg), HDLcholesterol (HDLc) and LDL-cholesterol (LDLc) fractions, and theratio TC/HDLc. They used laboratory methods based on the enzyme
principle for both CT (Dimension AR, Dade Behring Inc., USA) andTg and HDLc (REflectron IV, Roche, Switzerland). The LDLc level
fraction was calculated according to Friedwald’s formula: LDL = CT
- (HDLc + Tg/5). Were considered normal: CT <200 mg%, Tg <150
mg% and HDLc> 45 mg% in males and > 50 mg% in females, LDLc
<115 mg%, CT/HDLc between 2 to 3.5 (7, 11, and 8).
RESULTS AND DISCUSSION
The group of children and adolescents was represented by
83 subjects, aged 7-17 years (Tab. II). All children take in the
study had type 1 diabetes.
Table II. Distribution by age and sex of children and adolescents group
Age

Number ofcasesnFemale

%

n

Male

%

n

%

0- 4 years

-

-

-

-

-

-

5-9 years

2

2.4

2

100

-

-

10–14 years

32

38.56

22

68.75

10

31.25

15–17 years

49

59.04

47

95.92

2

4.08

Dyslipidemia is one of the most common metabolic
abnormalities from diabetes mellitus and one of the mostimportant cardiovascular risk factors (in adulthood) present in46

this disease.
The normal lipid parameters are as follows (Tab. III):
Table III. The normal lipid parameters (16)Lipid parameters

Normal values

Total serum cholesterol (CT)<190 mg%

Serum triglycerides (Tg)<180 mg%

HDL-cholesterol (HDLc)>40 mg%

LDL-cholesterol (LDLc)<115 mg%

To assess the type of dyslipidemia was used European
Atherosclerosis Association classification, which divides themaccording to severity (Table IV):
Table IV. The classification of dyslipidemia according to European
Arteriosclerosis Association (16)Dyslipidemia typeClassification

Lipid fractions(mg %)

Hypercholesterolemia

mild
mediumsevereCT = 200-239
CT = 240-300
CT > 300

Hypertriglyceridemia

mediumsevereTg = 200-400
Tg > 400

Mixed hyperlipemia

mediumCT = 200-300
or Tg = 200 – 400
CT > 300
or Tg > 400

severeAccording to this classification, at the group of children takein the study we obtained the results presented in Table V.
Table V. Disturbances types of lipid metabolism in children and adolescents group

Dyslipidemia typeClassification Children and ado-

Hypercholesterolemia
mild
mediumsevereHypertriglyceridemia
mediumsevereMixed dyslipidemia
mediumsevereNormalAnother changes- decreased Tg- decreased CT- decreased Tg andCTlescents group(n = 83)
n
%
6
7.22
4.81
4
2
2.4
1
1.2
1
1.2
0
0
56
67.46
24.09
20
19
22.89
1
1.2
-

In the study group, on the first place were the normal
Fiziologia - Physiology  2011.21.2 (70)values (67.46%), followed by other changes in lipid metabolism(24.09%), then by hypercholesterolemia (7.22%) and by
hypertriglyceridemia (1.2%).
All the 7 people with lipid metabolism disorders werefemale. The glycemic balance was unsatisfactory in 4 cases(fasting blood glucose over 150 mg% and HbA1c over 8%).
The LDLc was over 160 mg% in 3 cases, and low HDLc wasfound in two cases.
In type 1 diabetes appear classic” diabetic lipemia” which
is characterized by hypertriglyceridemia (Tg = 500-1000 mg %)promptly reversed after insulin therapy and metabolic balance.
The hyperlipemic mechanisms involved are increased of
VLDL-Tg production with decreases HDLc, decreased lipoprotein
lipase (LPL) activity, increased lipolysis and free fatty acids (FFA).
These mechanisms are induced by insulin deficiency or insulin
resistance (rare in type 1 diabetes) (16).
Dyslipidemia does not appear in the case of balanced
metabolic type 1 diabetes. In the case of type 1 diabetes
unbalanced or at onset, are increase Tg and decrease HDLc,
eventually grow and LDLc. These changes form the panel ofhyperlipoproteinemia type IV (hypertriglyceridemia) and/or type
II B hyperlipoproteinemia (mixed hyperlipidemia).
A number of studies in young show that the prevalence ofdyslipidemia is rare among them. It is influenced primarily by
the level of glycemic control (10). A study in Spain showed thatthe increase in LDLc was found in 16% of cases, increase intriglycerides in 5% of cases and lower HDLc in 20% of cases. The
females’ persons were more likely predispose to the presence ofdyslipidemia. With metabolic balance occurs also the normalizingof lipid metabolism disorders (15).
The adult group consisted of 650 people, young adults andelderly adults, aged between 17 and 79 years (Table VI). The
subjects with diabetes mellitus, which in time present thyroid
disease, and subjects with thyroid disease who subsequently
present glycoregulation disorders or diabetes mellitus
represented this.
Table VI. Distribution by age and sex of adult group
Age

Number of
cases
n
%

Female
n

%

Male
n

%

18 – 19 years

11

1.7

10

90.9

1

9.1

20 – 29 years

29

4.46

27

93.1

2

6.9

30 – 39 years

48

7.38

43

89.58

5

10.42

40 – 49 years

168

25.84

141

83.93

27

16.07

50 – 59 years

219

33.7

209

95.43

10

4.57

60 – 69 years

118

18.15

112

94.91

6

5.09

70 – 79 years

57

8.77

46

80.7

11

19.3

The adult group was subdivided in function of the type ofglycemic balance in 4 subgroups (Fig. 1):
group with type 1 diabetes represented by 60 cases(9.23%)
2011.21.2 (70)  Fiziologia - Physiology

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 117 cases (18 %)Fig. 1. Distribution of cases by type of changes in glycemic balance

Dyslipidemia does not appear in the case of DM metabolicbalance. Dyslipidemia is considered diabetic if it is corrected
through optimizing glycemic control (diet, oral hypoglycemic
agents or insulin). In general, the characteristics of dyslipidemia
in DM are:represent 55-65% in unbalanced DM toward to 25%
in balanced DM and 20% in non-diabetic population
the hypertriglyceridemia is predominant toward to
hypercholesterolemia (as in the general population)
it is prevalent in type 2 diabetes (50-70%) toward totype 1 diabetes (20%) due to the combination of dyslipidemia
risk factors (14).
It is known that in severe thyroid deficiency appear thefollowing disorders of lipid metabolism:
the cholesterol is increased (especially LDL-cholesterol)the beta-lipoproteins are increased and the ratio beta/alpha-lipoprotein is decreased
hypertriglyceridemia
Intensity of hyperlipemic syndrome is directly proportional tothe degree of thyroid failure. This syndrome can be considereda sign of progression of the disease or of effectiveness of substitution therapy. In the case of subclinical hypothyroidism, it canworsen pre-existing dyslipidemia commonly seen in patients with
type 2 diabetes; as a result increases the risk of cardiovasculardisease. Adequate replacement therapy with thyroxin will correct
the lipid metabolism disorders. In the case of hyperthyroidism thecholesterol is always low. According to this classification, in thegroup of adults take in the study we obtained results presented
in Table VII.
Table VII. Types of changes in lipid metabolism in the adult group47

Dyslipidemia type/severity

DM type 1n%DM type 2 IGT
n %
n %

IFG
n %

HyperCT25

41.66

39 13.44

67

36.61

61 52.13

mild

16

26.66

31 10.68

38

20.76

21 17.94

medium

9

15

8

2.75

22

12.02

33 28.2

severe

-

-

-

-

7

3.82

7

5.98

2

3.33

21 7.24

12

6.55

3

2.56

medium2

3.33

18 6.2

12

6.55

3

2.56

severe

-

-

3

1.03

-

-

-

-

2

3.33

81 27.93

41

22.4

16 13.67

medium1

1.66

41 14.13

28

15.3

9

7.69

severe1

1.66

40 21.85

13

7.1

7

5.98

-

-

5

1.72

4

2.18

5

4.27

-

-

1

0,34

3

1.64

2

1.71

-

-

3

1.03

1

0.54

2

1.71

-

-

1

0.34

-

-

1

0.85

Hyper Tg

Mixed
HLP

Anotherchanges
Decreased
Tg
Decreased
CT
Decreased
Tg andCT

In the case of type 1 diabetes group, on the first place was
the normal values (51.66%), followed by hypercholesterolemia(41.66%), then by hypertriglyceridemia (3.33%) and by mixed
dyslipidemia (3.33%). Between TSH value and lipid fractions
was establish a weak, direct correlation (r = 0.10, p<0.001 forCT and r = 0.27, p<0.001 for Tg).
In the case of type 2 diabetes group, on the first place was
the normal values (31.03%), followed by mixed dyslipidemia(27.93%), then by hypercholesterolemia (13.44%) andhypertriglyceridemia (7.24%). Between TSH and lipid metabolism
has established a weak, direct correlation (r = 0.29, p<0.001 forCT and r = 0.03, p<0.001 for Tg).
In the case of IGT group, on the first place was
hypercholesterolemia (36.61%), followed by normal
values (32.24%), then by mixed dyslipidemia (22.4%) andhypertriglyceridemia (6.55%). Between TSH and lipid metabolism
has established a weak, direct correlation (r = 0.42, p<0.001 forCT and r = 0.27, p<0.001 for Tg).
In the case of IFG group, on the first place was
hypercholesterolemia (52.13%), followed by normal values(26.49%), and then by mixed dyslipidemia (13.67%) andother changes (4.27%). Between TSH - lipid metabolism hasestablished a weak, direct correlation (r = 0.13, p<0.001 for CT
and r=0.05, p<0.001 for Tg).
It is noted that at all types of changes in glycemic control
in the adult group predominate hypercholesterolemia and not
hypertriglyceridemia, this is likely due to the presence of thyroid
disease association.
A study in Kenya shows that in patients with type 2 diabetes
hypercholesterolemia were found in 70% of cases, while
hypertriglyceridemia only ≈ 30% of cases (13).
CONCLUSIONs
In the case of patients with diabetes mellitus associatedwith thyroid disease was an increase in the prevalence of lipid
48

metabolism disorders. Among all types of dyslipidemia foundthe prevalence of hypercholesterolemia and mixed dyslipidemia,
which shows a significant influence on thyroid hormone changes
on lipid metabolism disorders.
Regarding gender, dyslipidemia was more common in
women because the prevalence of thyroid disorders at them.
The thyroid disorders cause significant physiologicaleffects. Subclinical hypothyroidism may increase LDLc andaggravation of preexisting dyslipidemia, increasing thus the riskof atherosclerosis. Subclinical hyperthyroidism may increase
the risk of cardiac arrhythmias and may cause worsening of apreexisting ischemic heart disease. Because diabetic patientsare an increased risk for cardiovascular disease, early diagnosis
and treatment of subclinical thyroid disease is important.
The presence of antithyroid antibodies (antiperoxidasetype) allows early detection of autoimmune thyroid disorders,
particularly of the hypothyroidism. In those in whom these
antibodies are present, thyroid function should be investigatedfor early detection of any disorder to establish appropriate
treatment.
Since dyslipidemia are involved in the occurrence of
cardiovascular disease, early detection of patients with diabetes
mellitus and associated thyroid disease is necessary.
In the case of patients with type 1 diabetes is necessary
to determine antithyroid antibodies antiperoxidase type. If they
are present, annual monitoring is required for TSH as early
detection of hypothyroidism. If thyroid hypofunction has no major
implications for glycemic control, atherosclerotic complications of
myxedema adversely affect long-term prognosis of association
between diabetes mellitus and hypothyroidism. If they are absent,
it is necessary to determine TSH at an interval of 2-3 years.
In the case of patients with type 2 diabetes, TSH
determination is necessary at an interval of five years.
Also, to avoid the negative impact of dyslipidemia on the
cardiovascular system, it is necessary the correct balance ofdiabetes, the correct treatment of thyroid disorders associatedand the correct treatment of lipid metabolism disorders if theyoccur.
REFERENCES

1. Assmann G, Schulte H. Relation of high-density lipoprotein cholesterol
and triglycerides to incidence of atherosclerotic coronary artery disease(the PROCAM experience). Prospective Cardiovascular Munster study.
Am J Cardiol. Sep 15 1992; 70 (7): 733-7
2. Austin MA. Plasma triglyceride as a risk factor for coronary heartdisease. The epidemiologic evidence and beyond. Am J Epidemiol.,
1989; 129(2): 249-59.
3. Bainton D, Miller Ne, Bolton CH. Plasma triglyceride and high density
lipoprotein cholesterol as predictors of ischaemic heart disease in British men. The Caerphilly and Speedwell Collaborative Heart Disease
Studies. Br Heart J., 1992; 68(1): 60-6.
4. Bezafibrate Infarction Prevention Study. Secondary prevention byraising HDL cholesterol and reducing triglycerides in patients withcoronary artery disease: the Bezafibrate Infarction Prevention (BIP)
study. Circulation, 2000; 102 (1): 21-7.
5. Citkowitz E, Gambert SR, Talavera F, Shenker Y, Cooper M, Griffing

Fiziologia - Physiology  2011.21.2 (70)GT. Hypertriglyceridemia, e-medicine, 2005.
6. Diabetes Atherosclerosis Intervention Study. Effect of fenofibrate on
progression of coronary-artery disease in type 2 diabetes: the Diabetes
Atherosclerosis Intervention Study, a randomised study. Lancet. Mar
24 2001; 357 (9260): 905-10.
7. De Backer G. European Guidelines in Cardiovascular Disease Prevention in Clinical Practice. European Heart Journal, 2003, 24:1601-1610
8. Grundy MS, Brewer HB, Cleeman JI, Smith SC, Lenfant C. Definitionof Metabolic Syndrome. Raport of the National Heart, Lung and Blood
Institute/ American Heart Association Conference on Scientific Issues
Related to Definition.Circulation, 2004, 109: 433-438.
9. Haim M, Benderly M, Brunner D. Elevated serum triglyceride levels
and long-term mortality in patients with coronary heart disease: theBezafibrate Infarction Prevention (BIP) Registry. Circulation. Aug 3
1999; 100 (5): 475-82.
10. Ismail IS, Nazaimoon W, Mohamad W, Letchuman R, Singaraveloo
M, Hew FL, Shuguna C, Khalid BA. Ethnicity and glycaemic control aremajor determinants of diabetic dyslipidaemia in Malaysia. Diabet Med.,
2001; 18 (6): 501-8.
11. MacLean D. CINDI dietary guide, Copenhagen, WHO regional Officefor Europe, (document EUR / 00 / 5018028)
12. Manninnen V, Tenkanen L, Koskinen P. Joint effects of serum
triglyceride and LDL cholesterol and HDL cholesterol concentrations
on coronary heart disease risk in the Helsinki Heart Study. Implications
for treatment. Circulation. Jan 1992; 85 (1): 37-45.
13. Otieno CF, Mwendwa FW, Vaghela V, Ogola EN, Amayo EO. Lipid
profile of ambulatory patients with type 2 diabetes mellitus at Kenzatta
National Hospital, Nairobi. East Afr Med J., 2005; 82: S 173-9.
14. Pencea C, Ionescu-Târgovişte C. Modificări lipidice în diabetul
zaharat. În: Tratat de diabet Paulescu, Editura Academiei Române,
2004: 881-893.
15. Perez A, Wagner AM, Carreras G, Gimenez G, Sanchez-Quesada
JL, Rigla M, Gomez-Gerique JA, Pou JM, de Leiva A. Prevalence andphenotypic distribution of dyslipidemia in type 1 diabetes mellitus: effectof glycemic control. Arch Intern Med, 2000; 160 (18): 2756-62.
16. Sabău I, Mărăzan M. Prognosticul şi complicaţiile diabetului zaharat. În: Diabetologie pediatrică, teorie şi practică, Editura Marineasa,
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17. Şerban V.Medicină Internă vol. 2, Editura Excelsior,Timişoara, 1996.

PARTICULARITATILE MODIFICARILOR METABOLISMULUI LIPIDIC LAPACIENTII CU DIABET ZAHARAT SI AFECTIUNI TIROIDIENEREZUMAT

Dislipidemiile se caracterizează prin creşterea colesterolului plasmatic, a trigliceridelor sau a ambelor, precum şi prin scăderea
HDL-colesterolului. Ele pot fi primare(genetice) sau secundare. Diagnosticul acestora este de obicei biochimic, prin determinarea
colesterolului total, a trigliceridelor şi a diferitelor fracţiuni lipoproteice. Tratamentul tulburărilor metabolismului lipidic constă în
modificarea stilului de viaţă, exerciţiu fizic şi administrarea medicamentelor hipolipemiante. Principala consecinţă a dislipidemiilor
netratate corespunzător o reprezintă apariţia bolii cardiovasculare sau agravarea uneia preexistente.
Dislipidemiile secundare reprezintă afecţiuni frecvent asociate atât diabetului zaharat, cât şi afecţiunilor tiroidiene. Scopul acestuistudiu a fost de a determina principalele tipuri de tulburări ale metabolismului lipidic întâlnite la pacienţii cu diferite modificări aleechilibrului glicemic şi afecţiuni tiroidiene. Lotul general studiat a fost reprezentat de 733 cazuri, cu vârste cuprinse între 7-79 ani.
Lotul studiat a fost subîmpărţit după criteriul vârstei în 2 loturi: lotul de copii şi lotul de adulţi. S-au folosit parametrii clinici, imagistici,
biochimici, hormonali, imunologici.
Cuvinte cheie: diabet zaharat, afecţiuni tiroidiene, dislipidemie, hipercolesterolemie, hipertrigliceridemie, boală cardiovasculară

2011.21.2 (70)  Fiziologia - Physiology

49

BENEFITS OF THYMECTOMY IN MYASTHENIA GRAVISCRISANDA VÎLCIU1, BOGDAN ISTRATE2, CARMEN TATU3, CONSTANŢA BĂNICĂ1, ANACÂMPEANU1, CONSTANTIN POPA4
1
Clinical Institute Fundeni, Department of Neurology, Bucharest
2
Center of Molecular Physiology and Neurobiology, University of Bucharest
3
Physiology-Immunology Department, „Victor Babes” University of Medicine and Pharmacy Timisoara
4
National Institute of Neurology and Neurovascular Diseases, Bucharest

ABSTRACT

Myasthenia Gravis (MG) is an autoimmune disease, caused by a direct auto-antibodies attack at the level of postsynaptic membrane acetylcholine receptors of the neuromuscular junction, characterized by fluctuating weakness and fatigability of skeletal
muscles.
Thymectomy represents an important link in Myasthenia Gravis treatment, because in some patients has determined the remission and improving in others the frequency and the intensity of myasthenic bursts.
Key words: myasthenia, thymectomy, improvement, immune response.

INTRODUCTION
Myasthenia Gravis is a chronic autoimmune disorder of theacetylcholine receptor, which evolve in bursts, being determinedby an autoimmune conflict at the level of neuromuscular junction. The cause of Myasthenia Gravis is still unknown. Normal
synaptic transmission is interrupted by the auto-antibodies,
which blocks the functional AchRs. As a consequence, these
auto-antibodies together with other immunological effectors arecapable to destroy the receptors, finally resulting in low numberof available receptors (1).
Thymic myoid cells may be responsible for the autoimmune
response, in our view T cells being sensitized against myoid cells.
The significant cytokines activity was reported in several studies
and was founded in thymus of MG patients.
Physiopathology and Immunopathology
In brief, specific antibodies for acetylcholine receptors in
Myasthenia Gravis are: AchR, MuSK, Titin, Ryanodine, andRapsyn. Regarding myasthenia, in the 1980’s, much immunological research was centered on auto-antibodies. AchR specificantibodies were found to belong of polyclonal IgG, predominantly,
but not exclusively to IgG1 and IgG3 (2). They are capable toreact with different epitopes on the AchRs surface (3). A variable
and large proportion of the serum antibodies bind to the one
main epitope on each on the two-α subunits of the receptor, also
called the immunogenic active region (4). AchR rarely recognizes
recombinant or synthetic peptide fragments. When activated, thecomplement system (membrane attack complex), will cause thelysis of muscle cell membrane, and internalization of AchRs in themembrane, increasing in this manner their degradation rate.
Thymectomy
The right definition of thymectomy with regards on myas-

thenia gravis is the Keesey point of view, otherwise known as
Keesey’s algorithm (5).
“Thymectomy is recommended for those relatively healthy
patients whose myasthenic symptoms interfere with her personal
life, long enough from them to accept a major surgical intervention. Although it is expensive and invasive treatment, thymectomy
is currently the only treatment modality that offers any chance ofremission without medication. Potential benefit of thymectomy
diminishes with the age of patient and the natural involution of
the thymus. In addition, surgical risk increases with age. Theage at which the risk exceeds the potential benefit, must be
individualized for each patient”.
Indications for thymectomy• presence of thymoma, confirmed by CT, indication at
any age• generalized myasthenia without thymoma• corticotherapy for at least month before surgery• minimal myasthenic deficit (QMG score < 10 points)• respiratory capacity > 60
Surgery classes recommended by Myasthenia Gravis
Foundation of America for Myasthenia Gravis:• T1- Trans-cervical thymectomy: standard and extended• T2- Videoscopic thymectomy: classic (VATS- VideoAssisted Thoracoscopic Surgery) and extended (VATET-VideoAssisted Thoracoscopic Thymectomy)• T3- Trans-Sternal Thymectomy: standard and extended• T4-Trans-cervical and Trans-sternal Thymectomy (maximal)
Complications of thymectomy:
Immediate: pneumothorax / hemothorax; atelectasis bybronchus blockage with viscous mucus; hematoma / serohematic

Received May 24th, 2011. Accepted June 25th, 2011. Address for correspondence: Bogdan Istrate, MD, ������������������������������������������������������������
Department of Neurobiology and Molecular Physiology, University of Bucharest, Romania, e-mail: bogdan_ellis@yahoo.com

50

Fiziologia - Physiology  2011.21.2 (70)flooded in anterior mediastinum; pericarditis; cardiac rhythm
disorders; paresis of phrenic nerve or recurrent laryngeal nerve;
myasthenic crisis.
Late: prolonged angina pectoris; wound or sternum infection; mediastinitis; thymic relapse can occur 5-10 years after
thymectomy - a period of improvement or remission of symptoms.
This was due to incomplete resection of thymus or persistentautoimmune thymic ectopia of mediastinal fat.
ThymomaThymoma is defined as a neoplasm of the epithelial-reticular
framework cells of the thymus. In the normal thymus, thesecells regularly displayed branching tonofilaments, maculae
adherents, elongated processes, and basal lamina. Thymomas
are composed of a mixture of neoplastic epithelial cells andnon-neoplastic lymphocytes, with proportion among them varying
widely from case to case and in different lobules of the same
tumor (6). Table I shows the morphological features of thymoma,
according with WHO classification, 2004.
Table I. Morphological aspects of thymoma(WHO classification, 2004)

cases (7).
About the oncogene proteins with a potential antigenic proliferation in thymoma, Bcl-2 oncogene reveals several features that
could be taken in discussion. This oncogene Bcl-2 is responsible
for the mechanism that desensitizes thymocytes. On the other
hand, an interesting remark is the fact that this oncogene protein
may trigger the auto reactive thymocyte apoptosis, and its presence in the thymic medulla may be similar with the presence of
inflammatory cells, possible mimicking the inflammation of thelymph nodes.
It was already discussed that the development of Myasthenia Gravis in patients with thymoma is probably caused by thesensitization of developing T-cells against autologous muscle-likeantigens, including the musacrinc and the acetylcholine receptor,
exposed by the neoplastic tissue.
Immunohistological insights
According to Salakou (2001), the presence of apoptotic bodies in hyperplasic thymus and in the thymic carcinoma, may be
an important objective that could be investigate with regards onanatomo-pathology of myasthenic thymus (Figure 1 A and B).

Fig. 1. Apoptotic bodies in hyperplastic thymus (TUNEL-peroxidase 100X) (A)and apoptotic bodies in a thymic carcinoma (TUNEL peroxidase 400X) (B)(After Salakoa, 2001).

Myasthenia gravis is associated with thymic hyperplasia in
65% of clinical cases and thymoma in 10 % of them. Thymoma
is present in a percentage about 30-45% of myasthenia gravis
2011.21.2 (70)  Fiziologia - Physiology

The anatomo-pathological and clinical special evidence of
patients with Myasthenia Gravis and thymus involvement should
outline the following data: gender, age, Osserman classification,
duration of the disease prior to surgery (months), medication, andprevious plasmapheresis, original histology diagnosis (hyperplasia, atrophy, thymoma, thymic carcinoma, and degenerated
thymic lipoma), thymus size (cm3), thymus weight (gr).
Autoimmune myasthenia gravis can be variable in its clinicalcharacteristics. The clinical data of myasthenia gravis patients
subgroups include: Early-onset myasthenia gravis (<40 years,
anti-AChR antibodies, thymic hyperplasia), Late-onset myasthenia gravis (>40 years, normal thymus, anti-AChR antibodies,titin antibodies, ryanodine receptors antibodies), thymomatous
myasthenia gravis (thymoma, anti AChR antibodies, titin antibodies, ryanodine receptors antibodies, KCNA4 antibodies),
anti MuSK myasthenia gravis (anti-MuSK antibodies, normal
thymus), anti-AChR and anti-MuSK negative myasthenia gravis(sero-negative generalized myasthenia gravis, thymic hyperplasia, antibodies against clustered AChR-70% of clinical cases)
and Ocular myasthenia gravis (anti-AChR antibodies in 50%
51

clinical cases).
Anyway, individual clinical evidence studies focused on
thymectomy, have reported results that shows the validity andbenefits of thymectomy via surgical techniques. In comparative
studies there is not clear evidence yet in order to establish which
thymectomy technique is accurate, superior and benefic in the
management of myasthenia gravis and patient comfort.
CONCLUSION
Thymectomy represents an important link in the treatmentof Myasthenia Gravis because in some patients has determinedthe disease remission and in others has improved the bursts infrequency and intensity. A correct pre-thymectomy management
reduces the post-operator complications rate, the hospitalization
period and also the number of the adjacent costs. Thymectomy
combined with corticosteroid therapy is more beneficial in treating patients with non-thymomatous Myasthenia Gravis thancorticosteroid therapy alone.

REFERENCES

1. Kaminski, Henry J. Myasthenia Gravis and related disorders. Humana
Press, 2009.
2. Vincent A. Unraveling the pathogenesis of Myasthenia Gravis. Nat
Rev Immunol., 2002; 2: 797-804.
3. Tzartos SJ et al. Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor.
Immunol. Rev., 1998; 63: 89-120.
4. Tzartos SJ, Seybold ME, Lindstrom JM. Specificities of antibodies
to acetylcholine receptors in sera from myasthenia gravis patientsmeasured by monoclonal antibodies. Proc. Natl Acad. Sci. USA, 1982;
79: 188-192.
5. Keesey J. A Treatment Algorithm for Autoimmune Myasthenia in
Adults. Ann. N.Y. Acad. Sci., 1998; 841: 753-68.
6. Wang Xinyum et al. Histological Classification of Thymoma and Its
Relationship with Myasthenia Gravis and Clinical Stages of the Tumor.
The Chinese-German Journal of Clinical Oncology, 2005; 4: 5.
7. Lewis JE, Wick MR, Scheithauer BW, Bernatz PE, Taylor WF. Thymoma. A clinicopathologic review. Cancer, 1987; 60: 2727-43.

BENEFICIILE TIMECTOMIEI ÎN MIASTENIA GRAVISREZUMAT

Miastenia Gravis (MG) este o boală autoimună cauzată de atacul direct al autoanticorpilor asupra receptorilor pentru acetilcolină dela nivelul membranei postsinaptice a joncţiunii neuromusculare, caracterizată prin slabiciune fluctuantă şi fatigabilitate a muşchilorscheletici. Timectomia reprezintă o verigă importantă în tratamentul Miasteniei Gravis, deoarece la unii pacienţi a determinat remisia,
iar la alţii ameliorarea puseelor miastenice, ca frecvenţă şi intensitate.
Cuvinte cheie: miastenie, timectomie, ameliorare, răspuns imun

52

Fiziologia - Physiology  2011.21.2 (70)

EXPERIMENTAL NEUROPHYSIOLOGICAL ALTERATIONSCAUSED BY COMBINED NANO-MANGANESE EXPOSUREZSUZSANNA MÁTÉ*, ANDREA SZABÓ, EDIT PAULIK, ANDRÁS PAPP
Department of Public Health, University of Szeged, Hungary
This article is republished due to some printing errors occurring in Fiziologia-Physiology, Vol. 21, Nr. 1(69), 2011, pages 18-19.

ABSTRACTManganese (Mn) is a well known heavy metal, causing central nervous system damage after chronic exposure. To model inhalational
Mn exposure, we used an animal model system in which male Wistar rats were intratracheally instilled daily with MnCl2 (2.5 or 5.0
mg/kg bw.) dissolved in distilled water; or with nanosuspension of MnO2 (2.5 or 5.0 mg/kg bw.); or with the combination of 2.5 mg/kg
solute MnCl2 and 2.5 mg/kg nanosized MnO2, administered with a short delay. After the 5 weeks treatment period open field activityof the rats was tested and electrophysiological investigation was performed; spontaneous and stimulus evoked action potentials wererecorded from somatosensory, visual and auditory areas of the cortex. Mn could reach the CNS and caused significant alterationsin the spectral distribution of the ECoG bands, increased the latency of the evoked potentials and influenced the locomotor activityof the rats. The observed effects showed a kind of additive effect in case of the combined exposure.
Keywords: manganese, nanoparticle, intratracheal instillation, evoked potentials, rat

INTRODUCTION
Manganese (Mn) is an essential micronutrient, cofactorof enzymes (Mn-SOD, GS), but has toxic effects after chronic
overexposure. It is often used in industry: in dry cells, coatedwelding rods, steel alloys, chemical fertilizers or pesticides.
Technical applications of manganese (Mn) often results in human exposure, mostly due to inhalation of metal dust and fumes(ATSDR, 2008) causing mitochondrial dysfunction and leading
to oxidative stress and excitotoxicity in the neurons (Taylor etal., 2006). Inhibition of voltage-gated Ca-channels and disturbed
release of neurotransmitters are also typical of Mn intoxication.
Mn evolves its deteriorating effects manly on the central nervous system. Manganism, a chronic human neurological disorder
resembling Parkinson’s disease (Bowler et al., 2006) often occurs
among welders and miners exposed to Mn-containing aerosols(Dobson et al., 2004). These dusts and fumes, arising fromsoldering and welding contain Mn nanoparticles as well.
Inhalational Mn exposure may result in massive internal
doses, depending on the size of the inhaled particles. Microscopic
particles cannot pass the blood-brain barrier, but submicroscopic
particles (that is, nanoparticles, with dimension 100 nm andbelow) have high mobility within the organism, so these canhave direct access to the CNS (Oberdörster et al., 2000, 2005).
According to this, it is worth to pay more attention on the effects
of inhaled Mn on the nervous system and its dependence on
the different physicochemical forms. In this study we aimed to
explore the alterations in the CNS caused by solute and nanosized and their combinations.
MATERIALS AND METHODS
Adult male Wistar rats obtained from the Breeding Centre of

the University (300-320 g body weight at start) were housed in
an air conditioned room maintained at 22 ºC, with 12-hour light/
dark cycle (light on at 06:00) and free access to tap water andstandard rodent chow.
Animals were divided into seven groups (with 8 animalseach) and were treated intratracheally with MnCl2 dissolved
in distilled water (LD: 2.5 mg/kg b.w. MnCl2; HD: 5 mg/kg b.w.
MnCl2) or with MnO2 nanoparticles (nLD: 2.5 mg/kg b.w. MnO2;
nHD: 5 mg/kg b.w. MnO2). In Comb group, rats received 2.5 mg/
kg b.w. MnCl2 and the same dose of MnO2 with a short delay.
The MnO2 nanoparticles (NPs) were synthesized by a technique
combining ultrasonic and hydrothermal treatment (for details,
see Sárközi et al., 2009) at the Department of Applied Chemistry, University of Szeged Faculty of Science and Informatics.
MnO2 NPs were suspended in hydroxyethyl cellulose (swollen
in phosphate buffered saline) which was used as vehicle andthat the rats in vehicle control (VC) group were treated with. An
untreated control (Cont) group was also applied.
Intratracheal instillation (1 ml/kg b. w.) was carried out inbrief diethyl ether anaesthesia. The treatment lasted for 5 weeks,
performed once a day, 5 days per week.
Before the beginning of the treatment period and on the day
following the last instillation, the rats were put into an open field(OF) box to test their spontaneous horizontal and vertical motor
activity in 10 min sessions.
Electrophysiological measurement was done after anesthetizing the animals with urethane (1000 mg/kg b. w., i.p.). The
left hemisphere was exposed, the animals were put in a stereotaxic device, and silver electrodes were placed on the primarysomatosensory (SS), visual (VIS) and auditory (AUD) areas.
Spontaneous electrical activity (electrocorticogram, ECoG) was

Received 15 February 2011. Accepted 20 March 2011. Address for correspondence: Zsuzsanna Máté, University of Szeged, Faculty of Medicine, Department of
Public Health, Dóm tér 10., Szeged, Hungary, Tel. +36-65-545-119, Fax. +36-62-545-120, email: matezs@puhe.szote.u-szeged.hu

2011.21.2 (70)  Fiziologia - Physiology

53recorded from these sites simultaneously for 6 min, and therelative spectral power of the frequency bands was determined(Kandel and Schwartz, 1985).
Stimulus-evoked activity was then recorded via thesame electrodes. Somatosensory stimulation was done byelectric pulses given trough a pair of needles inserted into thewhiskery skin (3-4 V; 1000, 500, 100 ms repetition time). Visual
stimulation was performed by flashes (1 Hz) delivered by a highluminescence white LED directly into the contralateral eye of therat. For acoustic stimulation, clicks (1 Hz, 40 dB), were applied
into the ear of the rat. Fifty stimuli of each modality per rat wereapplied and the evoked activity recorded. After averaging, latency
and duration of the evoked responses was measured.
The body weight of the animals was regularly measured
during the 5 weeks of the experiment.
Statistical analysis was done by two-sample t-test.
During the whole procedure, the principles of the EthicalCommittee for the Protection of Animals in Research of the
University were strictly followed.
RESULTS
Mn administration caused significantly slowed body weightgain in all treated groups (vs. Cont), from the first week on (Fig.
1).

Fig. 2 Effect of MnCl2 and MnO2 treatment on the OF activity of the rats after
the treatment. (A:time B:count) *,**;*** p<0.05, 0.01, 0.001 vs. Cont; #, ##,### p<0.05, 0.01, 0.001 vs. VC.

Intratracheal instillation of MnCl2 and MnO2 significantly
altered the spectral distribution of the electrocorticogram. The
general trend of the ECoG band spectrum change was different
in each modality depending on the treatment type (solute or nanosized Mn). MnCl2 treatment had a strong effect on theta band andthe increase was significant in each cortical area (vs. VC). Effect ofhigh dose MnO2 treatment was mainly seen on the VIS and AUD
ECoGs. On the VIS ECoG, the proportion of the fast frequency
bands increased and that of the slow bands decreased. On thecontrary, spectral distribution of the AUD ECoG showed decreasein fast bands, and increase in slow bands. These changes weresignificant in case of gamma and delta bands (Fig. 3).

Fig. 1 Body weight gain. *,**;*** p<0.05, 0.01, 0.001 vs. Cont; #, ##, ###p<0.05, 0.01, 0.001 vs. VC.

The open field test after the 5 weeks treatment period
showed decreased motor activity in all the treated rats. Increased
immobility and decreased rearing was observed both in the MnCl2
and MnO2 treated groups. The time spent with local activity
increased and showed mild significance in HD, nHD and Combgroups (Fig. 2a). Changes in activity counts were similar to theabove mentioned alterations, confirming that the OF activity of
the rats shifted to hypomotility, especially in MnCl2 treated andComb groups (Fig. 2b).

Fig. 3 Frequency spectrum of the spontaneous cortical activity. (SS-somatosensory, VIS-visual, AUD-auditory area) *,**;*** p<0.05, 0.01, 0.001 vs. Cont;#, ##, ### p<0.05, 0.01, 0.001 vs. VC.

54

Fiziologia - Physiology  2011.21.2 (70)From the parameters of the cortical evoked potentials,
lengthening of the SS latency, caused by MnCl2 treatment,
was the most prominent. MnO2 treatment did not significantly
affect the SS latency, but in Comb group a combined effectof the two forms of Mn was seen (lower, but significant
increase; Fig. 4). SS amplitude was only affected in nLD
group, where the increase was significant. In case of fast
stimulation (10 Hz), the same trend was discovered, but
the effect of the combined treatment – strong decrease –appeared to be significant (Fig. 5). Between VIS and AUD
latency there was an opposite alteration, similarly to the
ECoG findings (Fig. 6).

Fig. 4 Comparison of the latency of somatosensory evoked potentials of thetreated groups. *,**;*** p<0.05, 0.01, 0.001 vs. Cont; #, ##, ### p<0.05, 0.01,
0.001 vs. VC.

cal parameters the applied animal model proved to be suitablefor intratracheal Mn intoxication follow up. As it was indicated inprevious experiments (9), Mn had access to the CNS and theobserved electrophysiological alterations were caused by Mn
accumulation in the brain.
In the regulation of OF activity, which is known to be affectedby Mn (Normandin et al., 2004), dopaminergic structures play
important role (Alexander et al., 1990). Alterations in corticalevoked activity due to Mn can be explained by Mn-dependent
inhibition of astrocytic glutamine synthetase (�����������������
10)��������������. Desensitization and slowed action of the thalamocortical afferents (due toenhanced glutamatergic transmission) might cause lengtheningof the latency of the cortical evoked potentials. On the otherside, nerve conduction can be damaged as well. Mn might alsointerfere with Ca channels (11) and with mitochondrial energy
production (12).
Decrease of slow (delta, theta) and increase of fast (beta,
gamma) bands of the ECoG (or the opposite in VIS-AUD ECoG)
in the same rats might have resulted from impaired collateral
input of the glutamatergic afferents. Altered EEG and eventrealated potentials, which was found in this experiment in VIS
and AUD recordings, were also described in case of humanoccupational Mn exposure.
The results further suggested that electrophysiological tests
might be more sensitive to the effects of Mn than general toxicological or neurobehavioral ones, which is potentially relevant
both in experimental work and in hygienic toxicology.
REFERENCES

Fig. 5 Comparison of the amplitude of somatosensory evoked potentials of
the treated groups. *,**;*** p<0.05, 0.01, 0.001 vs. Cont; #, ##, ### p<0.05,
0.01, 0.001 vs. VC.

Fig. 6 Comparison of the latency of visual and auditory evoked potentials of
the treated groups. *,**;*** p<0.05, 0.01, 0.001 vs. Cont; #, ##, ### p<0.05,
0.01, 0.001 vs. VC.

DISCUSSION
According to the observed changes in the electrophysiologi2011.21.2 (70)  Fiziologia - Physiology

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55

11. Büsselberg D. Calcium channels as target sites of heavy metals.
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dysfunction and DNA fragmention in rat primary striatal neurons. Brain
Res. Bull., 2001; 15: 225-228.

ALTERARI NEUROFIZIOLOGICE EXPERIMENTALE DETERMINATE DEEXPUNEREA COMBINATA LA NANOPARTICULE SI MANGANREZUMAT
Manganul (Mn) este un metal greu bine cunoscut, care determina injurii la nivelul sistemului nervos dupa expunerea cronica. Pentru
a crea un model de expunere inhalatorie la Mn, am folosit un model animal, in care sobolanii masculi Wistar au fost instilati zilnic
intra-traheal cu MnCl2 (2,5 sau 5,0 mg/kg corp), dizolvata in apa distilata, sau cu nanosuspensie de MnO2 (2,5 sau 5,0 mg/kg corp);
au fost folosite si combinatii de 2,5 mg/kg solutie MnCl2 si 2,5 mg/kg MnO2 in suspensie de nanoparticule, care au fost administrateconsecutive, intr-un interval de timp scurt. Dupa o perioada de 5 saptamani de tratament, animalele au fost testate din punct devedere al activitatii si s-au efectuat investigatii electrofiziologice; au fost inregistrate potentiale de actiune evocate si spontane la
nivelul ariilor corticale somato-senzoriale, vizuale si auditorii. Mn a ajuns la nivelul SNC si a determinat alterarea semnificativa aspectrului de distributie al benzilor ECoG, a indus latenta potentialelor evocate si a influentat activitatea locomotorie la sobolani.
Efectele observate au aratat un efect aditiv in cazul expunerii combinate la ambele solutii.
Cuvinte cheie: mangan, nanoparticlule, instilare intra-traheala, potentiale evocate, sobolan

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