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Fiziologia - Physiology  2009.19.2(62)CONTENTS
1. The Physiology of English as a Lingua Franca in Medicine ................................................................................................................................4
Iulia Cristina Frînculescu
2. Molecular Regulation of Skeletal Muscle Atrophy . ..........................................................................................................................................7
Carmen Tatu, Gabriela Tanasie, Daniela Puscasiu, Rf Tatu, Florina Bojin, Carmen Bunu
3. Tendencies in Smoking Status and Smoking Habits among Medical Students during the First Three Years of Medical Studies...........................12
Lavinia Noveanu, Florina Bojin, Ovidiu Fira-Mladinescu, Adriana Gherbon, Minodora Andor and Georgeta Mihalas
4. Establishment, Propagation and Maintenance of Primary Pulmonary Fibroblasts ..........................................................................................17
Bianca Matis, Carmen Bunu, Gabriela Tanasie, Calin Tatu, Florina Bojin, Luminita Cernescu, Carmen Tatu, Laura Marusciac, Virgil Paunescu
5. Dendritic Cells and T Cells Activation.............................................................................................................................................................21
Luminita Cernescu, Janina Jiga, Lucian Jiga, Carmen Bunu, Calin Cernescu, Bianca Matis, Florina Bojin, Carmen Tatu, Gabriela Tanasie, Virgil Paunescu
6. Correlation of Anthropometric Parameters with Age and Sex in Post Puberty.................................................................................................24
Bagiu Radu, Rotaru Iulia
7. Lycopene Involvement in Hepatic Lipid Disorders Due to Hyperthyroidism in Rats...........................................................................................27
Adela Elena Joanta, Sanda Andrei, Nicoleta Decea, Adriana Muresan, Remus Moldovan, Olivera Stanojlović, Dragan Djuric, Manole Cojocaru
8. Laryngeal Papillomatosis Management........................................................................................................................................................31
Delia Horhat, Raluca Mocanu, Marioara Poenaru, S. Cotulbea, Olivia Toma, L. Fara, C. Sarau, F. Horhat, N. Balica, Mihaela Prodea
9. Urban Noise and Environmental Complaints in Timisoara...............................................................................................................................37
Ernest Putnoky
10. BOOK REVIEW: Stressology, Adaptology and Mental Health - Daniela Motoc.................................................................................................39

CUPRINS
1. Fiziologia englezei ca lingua franca în medicină ................................................................................................................................ 4
Iulia Cristina Frînculescu
2. Reglarea moleculara a atrofiei musculaturii scheletice...................................................................................................................................... 7
Carmen Tatu, Gabriela Tanasie, Daniela Puscasiu, Rf Tatu, Florina Bojin, Carmen Bunu
3. Tendinte in statusul si obiceiurilor fumatorilor in cadrul grupului de studenti la Medicina din anul trei . .......................................................... 12
Lavinia Noveanu, Florina Bojin, Ovidiu Fira-Mladinescu, Adriana Gherbon, Minodora Andor and Georgeta Mihalas
4. Stabilirea, propagarea si mentinerea culturii primare fibroblastice................................................................................................... 17
Bianca Matis, Carmen Bunu, Gabriela Tanasie, Calin Tatu, Florina Bojin, Luminita Cernescu, Carmen Tatu, Laura Marusciac, Virgil Paunescu
5. Celulele dendritice si activarea limfocitelor T.................................................................................................................................................. 21
Luminita Cernescu, Janina Jiga, Lucian Jiga, Carmen Bunu, Calin Cernescu, Bianca Matis, Florina Bojin, Carmen Tatu, Gabriela Tanasie, Virgil Paunescu
6. Corelatii intre parametrii antropometrici, varsta si sex in perioada post-pubertate.......................................................................................... 24
Bagiu Radu, Rotaru Iulia
7. Efectul licopinei asupra tulburarilor lipidelor la nivel hepatic induse de hipertiroidism la sobolani .................................................................. 27
Adela Elena Joanta, Sanda Andrei, Nicoleta Decea, Adriana Muresan, Remus Moldovan, Olivera Stanojlović, Dragan Djuric, Manole Cojocaru
8. Managementul papilomatozei laringiene........................................................................................................................... 31
Delia Horhat, Raluca Mocanu, Marioara Poenaru, S. Cotulbea, Olivia Toma, L. Fara, C. Sarau, F. Horhat, N. Balica, Mihaela Prodea
9. Zgomotul urban si sesizarile legate de protectia mediului in municipiul Timisoara............................................................................. 37
Ernest Putnoky
10. RECENZIE DE CARTE: Stresologie, Adaptologie si Sanatate Mintala - Daniela Motoc......................................................................................... 39
2009.19.2(62)  Fiziologia - Physiology

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THE PHYSIOLOGY OF ENGLISH AS A LINGUA FRANCAIN MEDICINEIULIA CRISTINA FRÎNCULESCU
Department of Foreign Languages and Romanian“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT
Based on the theoretical research on the concepts of cross-linguistic influence and interference, the study outlines the effects of cross-linguistic influenceof English on Romanian, especially at the lexico-semantic level, in the scientific field of medicine. The analysis is pursued first diachronically, with focus on
the advent of English as a lingua franca in medicine, and finally synchronically, providing a short list of neological medical Anglicisms, and some samples oftexts in which lexical English borrowings occur. Notions such as language contact, bilingualism, positive and negative transfer are passed into review, andthe review closes on the necessity of a more thorough analysis of the present-day Romanian medical terminology.
Keywords: lingua franca, medical language, language contact, cross-linguistic influence, bilingualism

ENGLISH AS A LINGUA FRANCA IN MEDICINE– DIACHRONICAL ACCOUNT
In outline, physiology, as defined by one of the “giants in the fields of physiology and medicine”, Dr. Arthur Guyton, means “the science of function in livingorganisms” (9). My treatment of what I call “physiology of English” relies upon
analogy. It is a non-standard, own view of the topic, in which the functioning ofa language may be seen as resembling the functioning of the human body, or of
any organ of the human body.
A few words about the metalanguage would be of use now. The term lingua
franca refers to the earliest Romance-based pidgin and has gained the meaningof a widely used auxiliary language to enable communication between people ofdifferent mother tongues (13).
In medicine, English replaced, one by one, other lingua francas of communication. Latin was the lingua franca of Western medical writing for several centuries. The
roots of Western medicine lie in Greek. Medical learning was transmitted in Latintranslations of Greek and Arabic texts, mostly by translators whose first language
was not a European vernacular, but Arabic or Greek. Galen’s texts became available
in the XIIIth century in Latin commentaries, with several layers of additions. Medical
texts began to be translated into vernacular languages such as French, English, German, Portuguese, and Catalan in the XIVth and XVth centuries, almost simultaneously
in different parts of Europe. However, at that time, Latin retained its strong position
as a pan-European language of science. The situation started to change in France, atthe end of the XVIth century, and in England, at the end of the XVIIth century (14),when several authors began to publish in both vernacular languages and Latin.
But Latin still retained its position longer in other parts of Europe, for example inGerman-speaking countries. Several scholars think that publication in medicine fromthe XVIIth century onward played a part in nationalizing medical communication(13). In 1551, one of the first French medical dictionaries, Traicté familier des noms
grecs, latin, arabiques, ou vulgaires, aveques les définitions de toutes les maladies
qui surviennent superficiellement au corps humain, was published (11).
In the early XXth century, there were rival languages for the lingua franca position in science: French, German, and English. German was a very strong candidate
before the Second World War. German had served as a lingua franca in large parts ofEurope for centuries, for example, in the Baltic area since the Middle Ages, and thenames of several scholarly journals and series in many fields are still in German.
French medical literature, dictionaries and treatises were also at their peaktill the 1950s and 1960s, when the situation started to change in favour of English
with the increasing impact of Anglo-American culture. After the World War II, all
the countries in Western Europe were affected to a greater or lesser degree by thedominant role of the United States of America, which was related to several wellknown factors, such as their military, political, economic, scientific, and technological leadership, as well as the creation of the Atlantic Alliance and the diffusion of
the culture, lifestyles, and behaviours of the English-speaking world.
The contemporary status of English as a global language is primarily the resultof two factors: the former expansion of British colonial power, which peaked towards
the end of the XIXth century, and the emergence of the United States of America as
the leading power of the XXth century. It is the latter factor that continues to explain
the position of the English today, much to the discomfiture of some in Britain who
find the loss of linguistic preeminence unpalatable (7).
On the other hand, it is sometimes thought that English has achieved theworldwide status because of its intrinsic linguistic features. People have claimed
that it is inherently a more logical or more beautiful language than others, easierto pronounce, simpler in grammatical structure, or larger in vocabulary. This kind of
reasoning is naive, inadequate, there are no objective standards of logic or beauty tocompare languages, and questions of phonetic, grammatical, or lexical complexity

Received February 2009. Accepted May 2009. Address for correspondence: Cristina Frinculescu, Department of Foreign Languages and Romanian, “Victor Babes” Universityof Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, Timisoara 300041, Romania, e-mail: cristinafrinculescu@yahoo.com
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Fiziologia - Physiology  2009.19.2(62)are never capable of simple answers. For example, English may not have many
inflectional endings, but it has a highly complex syntax.
In the recent years, English has become a prime vehicle for the transmissionof information, which explains its nearly absolute dominance in most scientificfields, because not only the world’s most widely cited medical journals but also
most of the best contributions in science and medicine are published in English
in international European or American journals. To this overall presence of English
in traditional written communication systems, we must add the World Wide Web
and the computer networking Internet, whose predominantly English voice has
been rapidly exported from and imported into many languages. “Therefore, nonEnglish-speaking scientists, researchers, and practicing doctors have no otheroption but to learn English if they want to be informed of the latest developmentsin their fields” (1).
“As English has turned into the primary medium of international specialized
publication, many non-English-speaking scientists, being aware of the relevanceof medical literature in English to their work and wanting to obtain responses to it,
find it more effective to publish in English than in their native language” (1). In thisrespect, it is interesting to note that many nations, Romanian academic life included,
measure the productivity of their top international scientists and scholars by the
number of times their works are quoted in English-language publications with an
impact factor by the Science Citation Index.
Apart from being the primary medium of scientific publication, English has
likewise emerged as the main language of international meetings of specialists
and of international scientific exchanges. In fact, the high level of technical andscientific knowledge, the necessity of collaboration among several specialists inorder to establish a common base for work, and the complexity of the organizationof production and of services in today’s society are all factors that foster the use of
the same technical terms contemporaneously.
“This trend to increasingly use one lingua franca, and in relatively few journals
for each science, favours a smoother communication between scientists and,
consequently, a rapid progress in science” (1).
Thus, the continually increasing contact between non-English-speaking
scientists and the English-speaking scientific world, mainly through reading
and, to a lesser extent, through writing and attending conferences, reaches evennational meetings, everyday informal conversations between colleagues, andnational journals, such as the following Romanian medical journals: Physiology,
The Journal of Cardiovascular Surgery, Timisoara Medical Journal etc., in whicharticles are published directly in English.
However, despite the obvious advantages of the present-day status of English
as a lingua franca, its achievement nevertheless runs parallel to a series of interrelated pitfalls, of which we focus on the great number of loan words from English.

LANGUAGE CONTACT AND BILINGUALISM
One of the drawbacks entailed by the supremacy of English in the world of
science would be that the language contact between English and other vernacularlanguages brings about massive linguistic transfer, which could end up in altering
the cultural integrity of a language (8). In all cases of language contact, speakers
of one language may, deliberately or unconsciously, introduce into their language
features of another language to which they have been exposed. Language contact is
a process that determines the use of two or more languages, therefore, it is a sourceof individual or group bilingualism (3), or of multilingualism. Contact between
languages only happens in the minds of bilingual (or multilingual) speakers.
Nowadays, most of the world’s population is thought to be bilingual or
multilingual. Taking Europe as reference, many millions of Europeans are at least
2009.19.2(62)  Fiziologia - Physiology

bilingual, speaking both their mother tongue and the national language of thecountry they live in, and many of them can additionally speak a global language
or world language, like English, which is a good reason to believe that bilingualism or multilingualism has been the norm for most human beings at least for thelast few millennia.
On the one hand, language contact results in cross-linguistic influence or
language transfer, but on the other, language transfer is not always positive, on
the contrary, it may be negative, ranging from limited lexical borrowing with casual
contact and limited bilingualism to heavy structural influence from very intensive
contact and bilingualism.

ANGLICISMS IN THE ROMANIAN MEDICALLANGUAGE
Romanian medical terminology, which, as different from other non-English
European medical languages, seems to have been unduly overlooked in the recent
years, rests on a fundamentally Latin nomenclature and on neologisms built up
with roots, prefixes, and suffixes drawn from Greek and Latin, especially in thefields of anatomy and physiology. From the latter half of the XVIIIth century, when
Romanian medical writing appeared, to the recent past, Latin and French influenced
the Romanian medical language. Traces from other European languages, such as
Italian, and German may be found as well.
As English has turned into the most powerful medium of medical and scientificcommunication in Europe, it is not surprising to find many English words in Romanian medical language, which have also entered Romanian medical dictionaries(11), a proof of their acceptance within Romanian medical communities. We provide
a short list of neological Romanian medical Anglicisms, open to completion: banding, borderline, bridge to recovery, floppy, bridge to transplant, bypass, clubbing,pacemaker, end-stage, flail, flapping tremor, flutter, follow-up, graft, guideline,
patch, marker, rash, scallop, prick, pattern, screening, scratch, thrill, turnover, slice,
stripping, trigger, shunt, stem cell, target, feedback.
The lexemes of the above list are examples of univerbal lexical Anglicisms withgeminated vowels and consonants (eg. flutter, patch, pattern, scratch), multiverbal
lexical Anglicisms with consonant groupings (eg. feedback), compounds that
contain a noun and a particle, generally considered difficult to translate adequately(eg. follow-up, bypass, turnover), phrases with nouns related by a preposition, also
considered difficult to translate (eg. bridge to transplant, bridge to recovery), and–ing simple and compound terms, extremely popular with specialist writers (eg.
banding, screening, stripping) (1).
The influence exerted by English on the Romanian medical language has
affected all levels of linguistic systems, ranging from lexis and semantics to syntax
and pragmatics, with the borrowing of vocabulary items being nevertheless by
far the most common.
As previously stated, language transfer may be positive, when the loan-words
occur in response to a demand for the expression of a new concept originating inanother country, and the word/phrase adopted fits the phonetico-phonologicaland lexico-semantic Romanian environment, but also negative. Without a disregard
for the former type of cross-linguistic influence, a thorough survey of the latter isalmost compulsory for the medical language, where any terminological ambiguity
or error, affecting the oral code, as well as the written discourse, may have serious consequences in real life. Instance the following different cases of negativetransfer: false friends (Engl. dramatically - Rom. dramatic; Engl. murmur - Rom.
murmur; Engl. insult - Rom. insultă etc.), polysemantic words (switch, cleft, marker,
management), inadequate calques, either lexical or grammatical (Engl. in the
population - Rom. în populaţia), and English doublets (synonymous variants) for5

CMYKalready existing words in Romanian (Engl. rash / Rom. erupţie; Engl. pacemaker /Rom. stimulator cardiac).
And finally, as lexical items only function in texts, the following samples oftexts, taken from a national medical journal, the Romanian Journal of Cardiovascular
Surgery (4), in which the majority of the articles are written in English, with a few
exceptions, reflect the present state of the Romanian written discourse. The main
terms are introduced in italic and bold letters:
1. Ecocardiografia a devenit metoda de neînlocuit în evaluarea valvulopatiilor:
toate guideline-urile pentru valvulopatii folosesc numai criterii ecocardiografice înalgoritmul de decizie (5).
2. Valva trebuie considerată, metaforic vorbind, ca o structură dinamică,formată din şase cuspide dispuse în perechi de câte două scallop-uri, scallop-ul A1
vs P1, scallop-ul A2 vs scallop-ul posterior P2 şi perechea A3-P3 (5).
3. Sunt posibile şi alte soluţii, deşi sunt mult mai rar folosite pentru acest tip deleziune – folosirea de corzi artificiale de goretex pentru restabilirea coaptării valvei
la nivelul scallop-ului sau a tehnicii “edge to edge” […] (5).
4. Sunt deja clare astăzi atât ecocardiografiştilor cât şi chirurgilor conceptele devalvă mitrală floppy, de valvă mitrală flail, ca şi de prolaps valvular mitral (5).
5. Corecţia chirurgicală anatomică (switch arterial) a transpoziţiei de vase maria devenit în ultima decadă intervenţia de elecţie în ţările dezvoltate […] (6).
6. Am analizat datele pre-, peri- şi postoperatorii a 1461 pacienţi care au
fost supuşi unor operaţii primare de bypass aortocoronarian (on şi off pump)[…] (2).

A WORD IN CLOSINGThe present article, as incomplete and open to further discussions andindividual viewpoints, claims, and suggestions it may be, points out that the
linguistic material the present-day Romanian medical literature provides us withis at least interesting, if not fascinating to survey, and a linguistic study would be
extremely helpful, not to say absolutely necessary for a proper assimilation of thenumerous Anglicisms to the Romanian linguistic rules. English medical terms
borrowed should be at first well understood, in their phonological, semiotic andmorphosyntactic behaviour, and then adapted to our language, with a maximumof precision, clarity, and accuracy.
Without a close survey from linguists, Romanian medical terminology is on
the way of becoming more and more heterogeneous, a mixture of Hellenisms, andEnglish neologisms, and medical discourse, written and oral, in a constant effort tokeep up with the English medical language, is adopting mechanically/ad litteram
English words, phrases or grammatical structures, thus giving rise to dangerous
ambiguities and confusions.
The study of language, as Ferdinand de Saussure clearly defined it, in his
celebrated langue−parole dichotomy, is the study of life in so far it is the studyof society (12). That is why I imagined a parallel between a linguistic aspect, thefunctioning of language, and the functioning of the living organisms. Therefore,
to conclude this article in the same spirit it started, I will quote from Dr. Guyton’saddress to the American Physiological Society in 1975, appropriately entitled
Physiology, a Beauty and a Philosophy: “What other person, whether he be atheologian, a jurist, a doctor of medicine, a physicist, or whatever, knows morethan you, a physiologist, about life? For physiology is indeed an explanation oflife. What other subject matter is more fascinating, more exciting, more beautiful
than the subject of life?” (10).
In its turn, medical terminology, with all the linguistic and extra-linguistic
aspects entailed, offers both terminologists and medical practitioners a large andextremely beautiful field of research. However, as a Danish physician says,“there is norecognized discipline called medical linguistics, but perhaps there ought to be one”,
as the study of the language of medicine may be a challenge for linguists and doctors, offering to the latter a new dimension to their professional language (15).
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1. Alcaraz Ariza MA, Navarro F. Medicine: Use of English, in: Brown
K (ed): Encyclopedia of Language and Linguistics, second edition,
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2. Aszalos A, Sculeanu R, Oprea A et al. Factori predictivi ai
morbidităţii neurologice după chirurgia coronariană. Romanian
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Dicţionar de ştiinţe ale limbii, Bucureşti, Ed. Nemira, 2005.
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Romanian Journal of Cardiovascular Surgery 2006: 132-139.
6. Chira M, Opriţa S, Aszalos S et al. Transpoziţia de vase mari – tratament în perioada neonatală. Romanian Journal of Cardiovascular
Surgery 2006: 191.
7. Crystal D. The Cambridge Encyclopedia of the English Language,
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8. Eyraud D. Bilan d'une décennie. META 1974; 19, no. 1: 13-27.
9. Guyton AC, Hall JE. Textbook of Medical Physiology. Elsevier
Saunders, Philadelphia, 2006.
10. Guyton AC. Past-President’s Address. Physiology, a Beauty anda Philosophy. The Physiologist 8: 495–501, 1975.
11. Rusu V. Dicţionar medical, ediţia a III-a revizuită şi adăugită,Bucureşti, Editura Medicală, 2007.
12. Saussure F. Cours de Linguistique générale, in: Bailly C, Séchehaye
A (ed.), Paris, Éditions Payot, Grande Bibliothèque Payot, 1995.
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edition, Elsevier Ltd., 2006: 643-644.
14. Webster C. The Great Instauration: Science, Medicine and Reform
1626–1660, London, Duckworth, 1975.
15. Wulff HR. The language of medicine. Journal of the Royal Society of Medicine 2004; 97(4): 187–188.

FIZIOLOGIA ENGLEZEI CA LINGUA FRANCA ÎN MEDICINĂREZUMAT

Având la bază studiile teoretice asupra conceptelor de influenţă şi interferenţă lingvistică, articolul urmăreşte influenţa limbii engleze asupra limbii române
din domeniul ştiinţific medical, în special la nivel lexico-semantic. Analiza, la început diacronică, cu accent pe impunerea limbii engleze ca lingua franca în
medicină, este urmată de o abordare sincronică, oferind o scurtă listă de anglicisme medicale neologice şi câteva fragmente de text în care apar împrumuturi
lexicale din limba engleză. Sunt, de asemenea, trecute în revistă conceptele de contact lingvistic, bilingvism, transfer pozitiv şi negativ, iar trecerea în revistă
se încheie cu afirmarea necesităţii unei analize mai profunde a terminologiei medicale româneşti actuale.
Cuvinte cheie: lingua franca, limbaj medical, contact lingvistic, influenţă lingvistică, bilingvism

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Fiziologia - Physiology  2009.19.2(62)MOLECULAR REGULATION OF SKELETAL MUSCLE ATROPHYCARMEN TATU, GABRIELA TANASIE, DANIELA PUSCASIU, RF TATU, FLORINA BOJIN,CARMEN BUNU“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACTThis review focuses on the most recent findings related to molecular regulation of muscle atrophy. Prolonged periods of skeletal muscle inactivity due to bed
rest, denervation, hindlimb unloading, immobilization, or microgravity can result in significant muscle atrophy. Skeletal muscle health involves maintenanceof an intricate balance between protein synthesis and degradation. Atrophy results from a perturbation in this intricate modulation of the various synthetic
and proteolytic pathways and very little is known about mechanisms involved in initiation of the imbalance Muscle atrophy in a range of conditions is thought
to be due to an increased expression of the ubiquitin-proteasome proteolytic pathway. Catabolic agents such as cytokines, proteolysis-inducing factor, andreactive oxygen species are causing an increased gene expression of proteasome subunits. Glucocorticoids cause activation of transcription factors possibly
through an increase in expression of myostatin.
Key words: muscle atrophy, ubiquitin-proteasome system, glucocorticoids, myostatin.

INTRODUCTION
Muscle is a highly plastic tissue able to adapt to changing functional demands. Increased load on muscle results in an increase in its mass or hypertrophy,
whereas unloading or disuse leads to a decrease in mass or atrophy. Exercise isa key regulator of muscle mass, as is nutrition (18). Muscular atrophy regularly
occurs as a consequence of immobilization or disuse after sports injuries. Immobilization is a frequently used treatment for musculoskeletal injuries despitewell-documented resulting muscle cell atrophy, intramuscular fibrosis, andloss of muscle extensibility, strength, and endurance (12). Prolonged periodsof skeletal muscle inactivity due to bed rest, denervation, hindlimb unloading,immobilization, or microgravity can result in significant muscle atrophy. Several
experimental models deal with muscle atrophy and are suitable for investigations of the underlying mechanisms of muscle atrophy. Strength loss is the mostevident response to atrophy. Muscle strength decreases most dramatically during
the first week of immobilization; little further weakening occurs later on. This isreflected in changes in the EMG of disused muscles and can also be observed inmuscle weight and size of muscle fibers (1). Slow muscles with predominantly
oxidative metabolism are most susceptible to atrophy as indicated by various
findings: slow muscle fibers show greater atrophy than fast fibers; their relativeand probably absolute number is decreased in atrophic muscles; in addition,
the oxidative enzyme content is most severely affected by disuse. Autophagic
activities probably play an important role in early stages of muscular atrophy. The
oxygen supply to disused muscle may be impaired, although myoglobin content
is increased in atrophic muscle. The complete loss of mitochondrial function
during the first days of disuse may be of etiological importance. The amount ofconnective tissue is increased in atrophic muscle and surrounding periarticular
tissue which may lead into a vicious circle of musculoskeletal degeneration. An
almost complete recovery from atrophy is possible, yet often the recovery phaseis much longer than the total immobilization period (1). The muscle atrophy ischaracterized as decreased muscle fiber cross-sectional area and protein content,
fiber diameter, reduced force, fatigue resistance, increased insulin resistance aswell as a slow to fast fiber type transition. Skeletal muscle atrophy attributableto muscular inactivity has significant adverse functional consequences (10).
While the initiating physiological event leading to atrophy seems to be theloss of muscle tension and a good deal of the physiology of muscle atrophy has
been characterized, little is known about the triggers or the molecular signaling
events underlying this process. Decreases in protein synthesis and increases inprotein degradation both have been shown to contribute to muscle protein loss
due to disuse, and recent work has delineated elements of both synthetic andproteolytic processes underlying muscle atrophy. It is also becoming evident
that interactions among known proteolytic pathways (ubiquitin-proteasome,lysosomal, and calpain) are involved in muscle proteolysis during atrophy. Factors
such as TNF, glucocorticoids, myostatin, and reactive oxygen species can inducemuscle protein loss under specified conditions. Also, it is now apparent that thetranscription factor NF-B is a key intracellular signal transducer in disuse atrophy.
Transcriptional profiles of atrophying muscle show both up- and downregulation of various genes over time, thus providing further evidence that there are
multiple concurrent processes involved in muscle atrophy. The decreases in protein
synthesis and increases in protein degradation rates account for the majority of
the rapid loss of muscle protein due to disuse (10, 22). Because different events
initiate atrophy in different conditions, it seems that the regulation of protein
loss may be unique in each case. In fact differences exist between the regulationof the various atrophy conditions, especially sarcopenia, as evidenced in partby comparisons of transcriptional profiles as well as by the unique triggering
molecules found in each case. By contrast, recent studies have shown that manyof the intracellular signaling molecules and target genes are similar, particularly

Received March 2009. Accepted May 2009. Address for correspondence: Dr. Carmen Tatu, Physiology Department, “Victor Babes” University of Medicine and Pharmacy
Timisoara, Eftimie Murgu Square No. 2A, 300041, Timisoara, Romania, e-mail: carmen.tatu@umft.ro2009.19.2(62)  Fiziologia - Physiology

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among the atrophies related to inactivity and cachexia (11).

SKELETAL MUSCLE ADAPTATIONS TO
INACTIVITY
Because skeletal muscle is the most abundant tissue of the human body, thedeceases in its mass possibly will profoundly impact the whole body metabolism
and ultimately lead to the development of lifestyle-related diseases (2). Marked
decreases in absolute and relative protein contents of skeletal muscles in early stagesof disuse is one of the most significant adaptations of this tissue to reduced tension,
resulting in fiber shrinking and weakening. Disuse-induced skeletal muscle atrophy
is accompanied by a whole-body negative nitrogen balance in humans during
spaceflight or bed rest (2, 17). In addition, hindlimb suspension or immobilization for 7 days significantly decreased the total RNA content and the α-actin andcytochrome c mRNA expression in the muscle in rats (2). Moreover, other studies
also revealed that the RNA-to-DNA ratio in atrophic muscle decreases considerably
in rats and human, indicating reduced capacity for protein synthesis (9). Thus, it canbe deduced that changes in protein turnover (concomitatnt upregulation of protein
degradation and downregulation of protein synthesis) in myofibers is one of the key
mechanisms that orchestrate adaptation of skeletal muscle to unweighting. The balance between protein synthesis and degradation is a critical determinant of muscle
cross-sectional area (18). Net protein synthesis results in greater myofibrillar content
which is accommodated in a larger myofibres. Significant myofibre hypertrophy
also requires an increase in the number of myonuclei so that a constant myonuclear
domain (volume of cytoplasm supported by a single nucleus) is maintained. In a
muscle, the ratio of DNA/protein is fairly constant (16). Myofibres are post-mitotic
cells, and their nuclei do not proliferate. New myonuclei are provided by a population
called satellite cells (18). These cells lie just under the basal lamina of myofibres,
and are normally found in a quiescent state. Once activated by exercise or muscle
damage, satellite cells proliferate and fuse with existing muscle fibres, thus providing
new nuclei for hypertrophy and repair. The absence of a satellite cell proliferative
response following γ-irradiation of the muscle limits hypertrophic gains. Protein
synthesis depends on the energy status in the muscle as it is an ATP-dependent
process, and therefore is also regulated by the AMP-dependent kinase AMPK (4,18). Treatment of rats with an AMPK-activating drug leads to a reduction in protein
synthesis accompanied by a decrease in activation of mTOR, p70S6K and 4E-BP1.
Protein degradation resulting from disease or disuse can be inhibited by AKT
activation. This occurs because AKT phosphorylates and thereby prevents nuclear
translocation of the FOXO family of transcription factors. FOXO1 and FOXO3 regulate
the expression of two ubiquitin protein ligases in muscle. Ubiquitin ligases linkubiquitin to proteins thereby targeting them for degradation by the ubiquitin–
proteasome, an ATP-dependent proteolysis complex. Another pathway of protein
degradation in skeletal muscle is autophagy, the bulk degradation of proteins andorganelles by lysosomal enzymes. The mechanisms responsible for the induction
and regulation of the autophagy programme are poorly understood but appear to
involve FOXO transcription factors as well, in particular FOXO3. Autophagy can beinhibited by AKT, but not rapamycin. Thus, FOXO3 controls the two major systems ofprotein breakdown in skeletal muscle, the ubiquitin–proteasomal and autophagic/
lysosomal pathways (15, 18). However, the hormonal and molecular regulators that
act in response to weightlessness or unloading, and ultimately bring about skeletal
muscle wasting, are not fully understood (2).
In an article of Haddad F (9), muscular atrophy (model of spinal cord isolation)
was associated with a reduced transcriptional activity (via pre-mRNA analyses) of
myosin heavy chain (MHC) and actin. In addition, there was an increased gene
expression of enzyme systems impacting protein degradation (calpain-1; plus
enzymes associated with polyubquitination processes) that could further contribute
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to the protein deficits in the spinal cord isolation muscles via degradative pathways.
IGF-I receptor and binding protein-5 mRNA expression was induced throughout the
15-day period of spinal cord isolation, whereas IGF-I mRNA was induced at 8 and
15 days. These responses occurred in the absence of an upregulation of translational
regulatory proteins (p70 S6 kinase; eukaryotic 4E binding protein to compensate
for the decreased protein translational capacity. These data demonstrate that: themolecular changes accompanying spinal cord isolation - induced muscle atrophy
are not necessarily the reverse of those occurring during muscle hypertrophy,
and the rapid and marked atrophy that defines this model of muscle inactivity islikely the result of multifactorial processes affecting transcription, translation, andprotein degradation. In conclusion, the atrophy process in response to spinal cord
isolation is heavily impacted by a reduction in the transcriptional activity of genes
encoding key sarcomeric proteins (actin and myosin) of the muscle. This response
lowers the mRNA substrate available for translation and is not offset by significant
increases in the functional activity of enzymes regulating protein translation
processes. Furthermore, the protein deficits appear to be exacerbated by increased
gene expression of enzymes postulated to be associated with disassembly of thecytoskeletal and myofibril framework, and ubiquitination of proteins targetingthem for degradation by the proteasomal machinery. These data suggest that the
rapid and marked atrophy associated with the spinal cord isolation model is likely
the result of multifactorial processes affecting gene transcription and of proteintranslation and degradation.
The work of Wang X (19) demonstrates that Merg1a channel function
participates in initiation of skeletal muscle atrophy in response to muscle disuseor cachexia by signaling an increase in ubiquitin proteasome pathway proteolysis.
Merg1 channel function is an initiating factor acting upstream of atrophy: Merg1
proteins are detected (day 4 of suspension) before the onset of significant atrophy(day 7 of suspension) genetic and pharmacologic attenuation of Merg1 channel
function prevents atrophy in hindlimb-suspended mice. These studies also demonstrate that the Merg1a splice variant is expressed in skeletal muscle, while Merg1b
is not detected, which strongly suggests that the Merg1 channel in this tissue iscomposed of Merg1a subunits only. Also, expression of Merg1a, and not Merg1b,
results in decreased muscle fiber size and increased ubiquitin proteasome pathway
activity in wt-bearing mice. Perhaps the more extensive Merg1a NH2 terminus isnecessary for up-regulation of the ubiquitin proteasome pathway. Further, our datastrongly suggest that Merg1a channel function is necessary to the atrophic process
because both expression of the dysfunctional Merg1a mutant and astemizole treatment (pharmacologic channel block) inhibit the decrease in fiber size induced bysuspension. Interestingly, although ubiquitin proteasome pathway activity is known
to function during atrophic remodeling of the heart, physiologically relevant levelsof Merg1 current is necessary for normal cardiac and are not likely to induce atrophy.
Perhaps expression of the Merg1b splice variant in heart is involved in this regulation.
Therefore, the functional consequence of Merg1 channel current conduction may be
determined by Merg1 channel subunit composition. In summary, Merg1a channel
function is an initiator of disuse- and cachexia-stimulated atrophy, acting upstreamof ubiquitin proteasome pathway proteolysis.
Skeletal muscle size is regulated by anabolic (hypertrophic) and catabolic(atrophic) processes (13). Hypertrophy in adult skeletal muscle is accompaniedby the increased expression of insulin-like growth factor-1 (IGF-1). IGF-I increases
the size of human myotubes whether treatment begins while myoblasts are still
proliferating or after proliferation has ceased (4, 18). IGF-I appears to regulate human myotube size by activating protein synthesis, inhibiting protein degradationand inducing fusion of reserve cells. During differentiation in culture, the majorityof cells exit the cell cycle and fuse, but there is always a small number of so-called
reserve cells that remain mononucleated. Fusion of a greater proportion of reserve
Fiziologia - Physiology  2009.19.2(62)cells increases the number of nuclei found within myotubes (fusion index) andthis will result in larger myotubes. The effect of IGF-I on reserve cell recruitment
appears to be indirect and to result from increased production of the cytokine interleukin-13 by treated myotubes. It remains to be demonstrated whether inductionof satellite cells fusion is induced by interleukin-13 in vivo and whether expression
on this cytokine in muscle is regulated by IGF-I. It is also unclear whether fusionof nuclei is a cause or consequence of activation of protein synthesis and cell size
increase. When IGF-1 was overexpressed in the skeletal muscle of transgenic micean increase in muscle size resulted. Furthermore, addition of IGF-1 in vitro to differentiated muscle cells promotes myotube hypertrophy, supporting the idea thatIGF-1 is sufficient to induce hypertrophy (13). Skeletal muscle atrophy, denoted bya decrease in muscle mass and fiber size, can be driven by such disparate stimuli as
denervation, immobilization, sepsis, cachexia, or glucocorticoid treatment. Atrophy
is characterized by increases in protein degradation processes, particularly the ATPdependent proteolytic ubiquitin-proteasome pathway. During atrophy, there is anincrease in ubiquitin-protein conjugates and increased transcription of componentsof the ubiquitin degradation pathway. A screen for genetic markers of atrophy
identified two genes that are up-regulated rapidly in multiple models of muscle
atrophy in vivo, including dexamethasone-induced wasting, which also show highly
muscle-specific expression. By studying both atrophy and hypertrophy conditions
simultaneously, the study establishes a new set of regulated genes, those transcriptsthat are not only perturbed by an atrophy stimulus but that are inversely regulated
during hypertrophy. This inversely regulated subset of genes would presumably
constitute an even more reliable set of genetic markers than genes simply regulated
by either atrophy or hypertrophy individually, since the ability to be regulated by the
opposing conditions makes the mRNA profile of this group of genes a barometer ofthe growth state of the muscle. From a clinical perspective, the finding that IGF-1 candominantly and inversely modulate key atrophy-induced helps to further validate
this pathway as a target for activation by anti-atrophy therapeutics (13).

THE ROLE OF HSP IN MUSCLE ATROPHY
In the paper of Broome CS (19), it is show that skeletal muscle aging is characterized by atrophy, a deficit in specific force generation, increased susceptibility
to injury, and incomplete recovery after severe injury. The ability of muscles ofold mice to produce heat shock proteins (HSPs) in response to stress is severely
diminished. Studies using HSP70 overexpressor mice demonstrated that lifelong
overexpression of HSP70 in skeletal muscle provided protection against damage andfacilitated successful recovery after damage in muscles of old mice. The mechanisms
by which HSP70 provides this protection are unclear. Aging is associated with theaccumulation of oxidation products, and it has been proposed that this may play amajor role in age-related muscle dysfunction. The cellular mechanisms underlying
this age-related decline are unclear, although considerable support exists for a role ofreactive oxygen species (ROS) in modulating the aging process (3, 19, 21). Changes
in markers of ROS production in skeletal muscle during aging have received someattention , although the functional effect of these changes has not been clearlyexamined. Skeletal muscle contractions result in an increased ROS generation (14,19), which can be potentially damaging. However, muscle cells have defense
systems that provide protection against an increase in the production of ROS. The
two major endogenous defense systems involved in this adaptation in muscle are
the antioxidant defense enzymes (including superoxide dismutase - SOD, catalase,and glutathione peroxidase) and heat shock proteins. Up-regulation of thesesystems occurs in muscle in response to increased ROS production via activation ofredox-responsive transcription factors. Nuclear factor- B (NF- B) and activator
protein-1 (AP1) transcription factors are involved in the up-regulation of antioxidant
2009.19.2(62)  Fiziologia - Physiology

enzymes such as SOD and catalase in response to oxidative stress (19), whereas HSP
expression in response to acute stress in eukaryotic cells is primarily regulated by thetranscription factor heat shock factor 1 (HSF1). In summary, the inability of musclesof old mice to produce HSPs after stress results in the accumulation of cellular oxidation products and that overexpression of HSP70 protects against the age-associatedincrease in ROS-mediated damage to cellular components and preserves the abilityof muscle cells to activate redox-responsive transcription after stress, which results
in protection against the development of age-related functional deficits.
In June 2009, Dodd SL (7), show that heat shock protein 25/27 (Hsp25/27)
is a cytoprotective protein that is ubiquitously expressed in most cells, and is upregulated in response to cellular stress. In nonmuscle cells, Hsp27 inhibits TNF-alphainduced NF-kappaB activation. During skeletal muscle disuse, Hsp25/27 levels are
decreased and NF-kappaB activity increased, and this increase in NF-kappaB activity
is required for disuse muscle atrophy. Therefore, the purpose of his study was todetermine whether electrotransfer of Hsp27 into the soleus muscle of rats, prior toskeletal muscle disuse, is sufficient to inhibit skeletal muscle disuse atrophy and NFkappaB activation. The 35% disuse muscle-fiber atrophy observed in nontransfected
fibers was attenuated by 50% in fibers transfected with Hsp27. Hsp27 also inhibitedthe disuse-induced increase in MuRF1 and atrogin-1 transcription by 82 and 40%,
respectively. Furthermore, disuse- and IKKbeta-induced NF-kappaB transactivation
were abolished by Hsp27. In contrast, Hsp27 had no effect on Foxo transactivation.
The conclusion of this study was that Hsp27 is a negative regulator of NF-kappaB
in skeletal muscle, in vivo, and is sufficient to inhibit MuRF1 and atrogin-1 andattenuate skeletal muscle disuse atrophy.
Carmeli E (2009) show that certain proteins such as matrix metalloproteinase-2(MMP-2) and heat shock protein 70(HSP-70) play a role during the degradation process (6). They hypothesized that tetracycline can be used to reduce tissuedegradation in skeletal muscles exposed to immobilization. The right knee of old rats
(20-months-old) was immobilized by a rigid external fixator (EF) device for 1, 2, 3 and4 weeks. Aqueous Tetracycline solution was administrated 3 times a week, following
2 days after the EF was constructed. Control group I was immobilized for 3 weeks,
did not receive tetracycline but did received saline injection, and control group II only
received tetracycline for 3 weeks. MMP-2 and HSP-70 protein and mRNA levels in thegastrocnemius and soleus muscles were analyzed at the molecular level by RT-PCR
and the protein level using SDS-PAGE gels and western blots. We have shown thatrats treated by Tetracycline reduce the MMP-2 expression and HSP-70. Theses changes
mainly occurred in type IIb and type IIa muscle fibers. Tetracycline administration has
beneficial effect on expression of enzymes involved in protein degradation. This may
suggest a protective effect on protein degradation during immobilization. The mechanisms by which lifelong overexpression of HSP70 may preserve muscle function in old
mice are unclear (5). It is suggest that dysfunction occurs in muscle and other cells asa consequence of the accumulation of oxidative damage to cellular components. HSPs
are known to provide protection against acute reactive oxygen species (ROS) mediatedcell damage, although the effects of increased HSP expression on changes in markers
of ROS production have not been examined. The inability of muscles of old mice toproduce HSPs after stress results in the accumulation of cellular oxidation products
and that overexpression of HSP70 protects against the age-associated increase in
ROS-mediated damage to cellular components and preserves the ability of muscle
cells to activate redox-responsive transcription after stress, which results in protection
against the development of age-related functional deficits (5).

SIGNALING MOLECULES INVOLVED IN
MUSCLE ATROPHY
Various signaling proteins have been studied for roles in regulating disuse
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implicated in the pathogenesis of muscle atrophy have revealed potential targets
for therapeutic interventions. However, an effective application of these therapies
requires a better understanding of the relative contribution of these pathways tothe development of muscle atrophy in distinct pathological conditions.

Ubiquitin-proteasome system in disuse
atrophy
There is evidence that loss of lean body mass is usually caused by activation of
the ubiquitin-proteasome proteolytic pathway in muscle, but the pathophysiologicaltriggers that accelerate protein degradation are controversial (20). Inflammation isoften suggested as a trigger because many illnesses causing loss of lean body mass
are associated with increases in circulating cytokines. However, inflammation canbe linked to insulin resistance because high levels of circulating TNF and possibly
other cytokines can cause insulin resistance. Another potential proteolytic triggerof muscle protein breakdown is a decrease in the responses to insulin or IGF-I. For
example, there is evidence that insulin deficiency causes muscle protein breakdownby activating the ubiquitin-proteasome proteolytic pathway in processes that
include transcription of genes encoding subunits of this system. This is relevant
because catabolic conditions that stimulate muscle protein degradation by theubiquitin-proteasome proteolytic pathway such as aging, acidosis, chronic kidneydisease, or acidosis are often associated with insulin resistance (20). The presence of
these complicating factors raises the question of whether insulin resistance by itself
will stimulate protein metabolism and, if so, by what mechanisms. An activation of
the ubiquitin–proteasome pathway has been reported in disuse muscle atrophy,
possibly by a passive–active mechanism. The process of substrate ubiquitination
involves the cooperative interaction of at least three classes of proteins termed
E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligating)enzymes. The activation of the noncanonical NFkB pathway, which involves p50
and Bcl-3, and is not induced by inflammatory cytokines, has also been reported
in experimental models of disuse atrophy. It is possible that reactive oxygen species(ROS) activate NFkB directly. ROS can also stimulate FOXO activity. This evidence
link the oxidative stress produced in the muscles during unloading and immobilization with the activation of the ubiquitin–proteasome pathway. Inhibition of theproteasome with agents available since 1994 has shown significant interferenceof muscle proteolysis in disuse muscle. In addition, there are significant increases
in the expression of components of both the process of ubiquitination and of themany proteasome subunits with disuse.
Myostatin is a member of the TGFβ family of signal transduction proteins thatnegatively regulates muscle mass in the adults by inhibiting muscle regenerationand is a strong negative regulator of muscle growth. The increase in muscle massobserved in myostatin-null animals predominantly results from an increase in thenumber of muscle fibers (hyperplasia). Myostatin has been isolated as the genemutated in cattle characterized by abnormal hypertrophy of the skeletal muscle(8). Similarly, myostatin-null mice display an increase in muscle mass relative tocontrol animals, and gene mutation that precludes myostatin expression has been
identified in humans and dogs showing a hyper-muscular phenotype. Knockout
or mutation of this protein produces animals with markedly enlarged muscles as aresult of hypertrophy and hyperplasia (10). Consistently, systemic overexpression of
myostatin in mice causes significant loss in muscle mass, and the effect is reversed
by follistatin administration (8). Thus, factors that interfere with myostatin activity
can be considered anabolic signals. Systemic administration of this negative growth
regulator leads to muscle wasting in mice, and treatment of cultured muscle cellswith recombinant myostatin has resulted in the loss of protein and reduced protein
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synthesis rates (10). Moreover, myostatin expression is increased in some types ofmuscle atrophy). Human immunodefiency virus (HIV)-infected men have shownhigher levels of serum myostatin, indicating that myostatin may contribute tocachexia-type atrophy.

Glucocorticoids
In skeletal muscle, glucocorticoids decrease the rate of protein synthesis
and increase the rate of protein degradation (10). Disuse atrophy is associatewith increases in circulating glucocorticoid levels. Moreover, the binding capacity of corticosteroids also was increased markedly with disuse atrophy, and so it
seemed plausible that glucocorticoids could be an important trigger. However,when adrenalectomized animals underwent unloading, with or without cortisol
treatment, atrophy still occurred (10). Importantly, treatment of unloaded rats
with an inhibitor of glucocorticoids, RU-38486, also did not inhibit disuse atrophy.
Thus glucocorticoids do not appear to be required for disuse atrophy. In the caseof cachexia, glucocorticoids seem to be a contributing factor to muscle wasting, inpart because rats treated with RU-38486 plus TNF showed reduced proteolysis, but
protein loss was not completely attenuated (10). Glucocorticoids have been shownto cause atrophy of fast-twitch or type II muscle fibers (particularly IIx and IIb) withless or no impact observed in type I fibers. Therefore, fast-twitch glycolytic muscles(i.e., tibialis anterior) are more susceptible than oxidative muscles (i.e., soleus) to
glucocorticoid-induced muscle atrophy. The mechanism of such fiber specificity is
not known. In skeletal muscle, glucocorticoids decrease the rate of protein synthesis
and increase the rate of protein breakdown contributing to atrophy.The severity andthe mechanism for the catabolic effect of glucocorticoids may differ with age. For
example, glucocorticoids cause more severe atrophy in older rats compared withyounger rats. Furthermore, glucocorticoid-induced muscle atrophy results mainly
from increased protein breakdown in adult rats but mostly from depressed protein
synthesis in the aged animals (1, 9, 10).
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REGLAREA MOLECULARA A ATROFIEI MUSCULATURII SCHELETICEREZUMAT

Acest articol se refera la cele mai recente descoperiri legate de reglarea moleculara a atrofiei musculare. Periodele prelungite de inactivitate ale musculaturii
scheletice datorate repausului la pat, denervarii, imobilizarii sau imponderabilitatii, pot duce la atrofie musculara semnificativa. Pastrarea sanatatii musculaturii
scheletice presupune mentinerea echilibrului intre sinteza si degradarea proteinelor. Atrofia apare ca urmare a perturbarii acestui echilibru si din pacate secunosc inca destul de putine date despre mecanismele care contribuie la initierea acestui dezechilibru. In cursul atrofiei musculare s-a constatat cresterea
expresiei sistemului proteolitic ubiquitin-proteozom. Agenti catabolici cum ar fi: citokinele, factorul care induce proteoliza, speciile reactive ale oxigenului
determina cresterea expresiei genelor subunitatilor de proteozomi. Glucocorticoizii determina activarea transcriptiei unor factori care probabil cresc expresia
miostatinei.
Cuvinte cheie: atrofie musculara, sistemul ubiquitin-proteozom, glucocorticoizi, miostatina.

2009.19.2(62)  Fiziologia - Physiology

11

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TENDENCIES IN SMOKING STATUS AND SMOKING
HABITS AMONG MEDICAL STUDENTS DURING THE FIRSTTHREE YEARS OF MEDICAL STUDIESLAVINIA NOVEANU1, FLORINA BOJIN1, OVIDIU FIRA-MLADINESCU2, ADRIANA GHERBON1, MINODORA ANDOR3 and GEORGETA MIHALAS1
1
Department of Physiology, University of Medicine and Pharmacy “Victor Babes”Timisoara
2
Department of Physiopathology, University of Medicine and Pharmacy “Victor Babes”Timisoara
3
Department of Medical Semiology II, University of Medicine and Pharmacy “Victor Babes”Timisoara

ABSTRACTThe purpose of our longitudinal study was to evaluate whether smoking status and smoking habits changed during the first three years of medical studies. A total number of 350 medical students (126 male and 224 female) were surveyed every year, between 2003 – 2006, using a questionnaire including
demographic details, data of smoking status and smoking habits evaluated by the Fagerström test for nicotine dependence (FTND). The results of our study
revealed that percentage of male subjects current smokers was significantly higher than the same parameter evaluated in female subjects only in the first (p= 0.03) and the second year of study (p = 0.04). The percentage increase of new smokers related with the year of study was significant in female subjects (p= 0.01). Severity of nicotine dependence, estimated using the FTND score, significantly increased every year of study, both in male subjects (p = 0.002), as
well as in female subjects (p < 0.001). Upward trend in tobacco use, particularly in women, is a reason for concern. The relatively less encouraging smoking
data among our medical students suggest the need to promote tobacco education and intervention efforts in this population segment.
Key words: cigarette smoking, medical students, Fagerström questionnaire

INTRODUCTION
The most important determinant of human health is the increase in tobacco
related mortality and disability. Tobacco related deaths have been projected toincrease from 3.0 million in 1990 to 8.4 million in 2020, which made tobacco thelargest single health problem at this time (1).
There are 80 - 90% of deaths from chronic obstructive lung disease attributed to tobacco, and smokers have 6 times the risk of contracting this disease
compared with non-smokers. Similarly, 80 - 85% of lung cancer deaths are
attributed to tobacco use, with smokers having 10 times the risk compared with
non-smokers (2).
Physicians who take their professional role seriously have the opportunity
and responsibility to act on various levels to combat smoking, acting as models,
educators, therapists, and antismoking advisers. It has been observed that doctors
who smoke tend to be more permissive, and are less inclined to advise their patients
against tobacco use, and adopt a passive attitude towards smoking (3).
A comprehensive education for physicians on the subject of smoking
dependence is imperative, and the best possible time for the training is when
they are students.
The smoking habits of medical students have only rarely been the objectof studies and the interventions in Romania. A review of the results obtained inother countries reveals certain trends in the evolution of the smoking habits ofthis population.

The purpose of our longitudinal study was to evaluate whether smoking
status and smoking habits changed during the first three years of medical schoolstudies.

MATERIAL AND METHODS
A total of about 350 medical students, 224 female (age 21 ± 1.9 years) and
126 males (age 22 ± 2.20 years), were surveyed every year, between 2003 – 2006,
using a questionnaire included: demographic details (name, age, gender), data ofsmoking status (current, new, former, never) and data of smoking habits evaluatedby Fagerström test for nicotine dependence (FTND).
Subjects who smoked daily were asked to rate themselves as current smokers,
those who had smoked for the first time in the previous 12 months as new smokers,
those who had not smoked in the previous 12 months or longer as former smokers, while those who had smoked < 100 cigarettes in their lifetime were asked toconsider themselves non or never smokers.
The FTND is a widely used and validated 6-item questionnaire to asses
severity of nicotine dependence using a score range from 0 to 10 (Table I) (4, 5).
FTND also provides a scale for nicotine dependence severity as low, medium andhigh dependence, and allows analysis of other characteristics related to smoking
behavior, which define severity of nicotine dependence. A score < 4 suggests a
low level of nicotine dependence, and a score > 6 usually indicates a high level

Received February 2009. Accepted May 2009. Address for correspondence: Dr. Lavinia Noveanu, Physiology Department, “Victor Babes”University of Medicine and Pharmacy
Timisoara, Eftimie Murgu Square No.2A, Timisoara 300041, Romania, e-mail: lavinianoveanu@yahoo.com
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Fiziologia - Physiology  2009.19.2(62)of nicotine dependence.
Table I. The Fagerström Test for Nicotine Dependence

QuestionsScore

1. How soon after you wake up do you
smoke your first cigarette?
ƒ within 5 min
ƒ 6 to 30 min
ƒ 31 to 60 minƒ after 60 min
2. Do you find it difficult to refrain fromsmoking in places where is forbidden?
ƒ Yes
ƒ No
3. Which cigarette would you hate most to
give up?
ƒ The first in the morningƒ Any other
4. How many cigarettes per day do you
smoke?
ƒ 10 or less
ƒ 11 – 20
ƒ 21 – 30
ƒ 31 or more

1
0

1
0

0
1
2
3

6. Do you smoke if you are so ill that you
are100%in bed most of the3.33
day?
4.14
ƒ Yes
2.38
4.76
ƒ 80%
No
32.53

1
0

2.33

1
0 7.14
34.92

Statistic analysis was performed using Excell Microsoft Office 2003 and EpiInfo 6
software.40%
The central tendencies of the variables were expressed as a mean (M), and the
60.95
56.61
64.14
dispersion ones as standard deviation(sd). In order to performthe statistic comparisonsthe„t”-Student
20%test, as well as the variance analysis (ANOVA), were used for continuous
variables, and the Chi – square (X2) test for categorical variables.The values achieved wereconsidered 0%
significant for p < 0.05.

RESULTSYear 2

Year 3

SmokingNever
Status Current
New
Form er
Distribution of smoking status’variables was determined on each year of our study,
both in male gender subjects, and in female gender subjects.
Fig.1. The pattern of smoking status on male
We can observe the general tendency
of increasing the percentage of current
group
smokers in the diagrams presented above, which follows each year of study, both in2009.19.2(62)  Fiziologia - Physiology

80%

3.33

4.14

2.33

4.76

2.38

7.14

27.77

32.53

34.92

100%
80%

60%

60%

60.95

64.14

40%

56.61

20%

20%

0%

0%
Year 1
Never

60%

Year 1100%

40%

5. Do you smoke more frequently during
the first hours after waking than during therest of the day?
ƒ Yes
ƒ No

27.77

3
2
1
0

male subjects (p = 0.22), and in female subjects (p < 0.001), respectively. Significant
differences between the percentage of male and female subjects current smokers wererevealed in the first (X2 = 4.26, p = 0.03) and the second year of study (X2 = 3.97, p =0.04), respectively.
In the third year of medical education, this difference became insignificant (X2 =0.13, p = 0.72), thus suggesting the progressive increase of female smokers percentage
along with each year of study.
Graphs showed in Fig 1 and Fig 2 also reveal the increasing percentage of newsmokers along with each year of study, both in male subjects (p = 0.36), and in female
subjects (p = 0.01), respectively. The highest rate of new smokers percentage increase
was showed in male subjects in the third year of study (X2 = 4.28, p = 0.03), and infemale subjects in the second (X2 = 9.46, p = 0.002) and in the third year of study (X2= 3.70, p = 0.05), respectively.
Considering the status of former smokers, we did not found significant differences
related to the year of study (p = 0.30) or subjects’gender (X2 = 1.91, p = 0.16 for the firstyear; X2 = 0.69, p = 0.40 for the second year; X2 = 3.70, p = 0.05 for the third year).

Year 2Current

Year 3

New

Form erN

Fig. 1. The pattern of smoking status on male group

Fig.2.

Fig.1. The pattern of smoking status on malegroup
Severity of nicotine dependence
Severity of nicotine dependence was estimated using Fagerström score, which
4.33

100%

4.46

80%

18.32

60%
40%

72.89

7.14

4.33

10.26

10.26

22.76

33.03

59.80

52.38

20%
0%
Year 1
Never

Year 2Current

New

Year 3FormerFig. 2. The pattern of smoking status on female group

Fig.2. The pattern of smoking status on female
group
proved to have a higher significance when analyzing the male subjects, compared

13

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with female subjects, during all years of study (Tab.II).
 of Fagerström score together with
 was also revealed
Significant
increase%
100year of study, both in male subjects (p = 0.002), as well as in female
the next- g- - subjects80(p < 0.001). 68.29
63.63

100
60

Table II.- FTND
- Nicotine dependence
score score
Table II.
FTND
Nicotine
dependence
36.58

40

Year 19.51
20

Male

28.3
15.920.45 Female
13.2

5.19 r 1.85

Year 2Year
1 r 1.95 Year5.25
2
6.35
r 2.17Year 3 0.006

Year 2

6.51 r 2.05

0

Low

80
60
40
20

%


- g-

High





68.29

- -

36.58
15.920.45

19.51

-

63.63

56.6

28.3
13.2

Year 1

Year 2
Low

MediumYear 3High

Fig.Fig.
3. The3.pattern
nicotine dependence
severitydependence
on male group (#p<0.001,
Theofpatternof nicotinef&p=0.009,
p=0.002).
severity on male group(p < 0.001, gp = 0.009, p = 0.002).

CMYK









50.98

44.59

35.29
13.72

28.37
27.02

44.32
37.3
28.86

0
Year 1

Year 2
Low

MediumYear 3High

Fig. 4. The pattern of nicotine dependence severity on female group (#p<0.05,
Fig. 4. The patternfp<0.001).
of nicotine dependence

severity on female group
III), as well in female subjects
in Tab.III and Tab.IV show
0.05, gDatap <presented
0.001).
(p –<(Tab.IV).
Table III. FTND
intermediarypoints for male group
significant change of smoking behavior along with each year of study only in caseof female subjects.
Q
Year 1
Year 2
Year 3p
Table III. FTND – intermediary points for male group(n = Table41) III. FTND
(n–=intermediary44)(n for= 53)
pointsmale group

Q1.
1.
2.
3.
4.
5.

2.
3.
4.
5.
6.

Year
1
2.42
r 0.96(n = 41)0.50 r 0.50
2.42 r 0.96
0.69 r 0.46
0.50 r 0.50
1.14 r 0.52
0.69 r 0.46
0.80 r 0.40
1.14 r 0.52
0.30 r 0.46
0.80 r 0.40

2
r 0.93
2.40Year(n = 44)
0.35 r 0.48
2.40 r 0.93
0.63 r 0.49
0.35 r 0.48
1.04 r 0.67
0.63 r 0.49
0.82 r 0.38
1.04 r 0.67
0.33 r 0.47
0.82 r 0.38

2.44Yearr 0.793(n = 53)
0.29 r 0.46
2.44 r 0.79
0.62 r 0.48
0.29 r 0.46
1.16 r 0.72
0.62 r 0.48
0.81 r 0.39
1.16 r 0.72
0.35 r 0.48
0.81 r 0.39

0.96p
0.11
0.96
0.82
0.11
0.62
0.82
0.98
0.62
0.91
0.98

The results are expressed as M r sd
0.91
0.47
0.35dependence
r 0.480.30 rof
0.46
0.33 rtest
Q6.= question
Fagerström
of nicotine(FTND)The results are expressed as M r sd
The results are expressed as M ± sd
Q = questionoftest ofnicotine
Q = question ofFagerström
Fagerström test of nicotine
dependence(FTND)dependence (FTND)
Table IV. FTND
- intermediary points for female group
Table IV. FTND - intermediary points for female groupQ
Year
1
Year 2
Year
p
Table
IV.
FTND
- intermediarypointsfor3female group(n = 51)(n = 74)(n = 97)
Year 1
Year 2
Year 3p<0.001
1.94 r 1.17
1.36 r 0.97
1.38 r 0.92
(n = 51)(n = 74)(n = 97)0.01
2.
0.58 r 0.49
0.40 r 0.49
0.55 r 0.49
<0.0011.
% r 0.97
1.94 r 1.17
1.36
1.38 r 0.92



100
<0.001
3.
0.46 r 0.50
0.17 r 0.380.14 r 0.35
0.01
2. 800.40 r 0.49
0.58 r 0.49
0.55 r 0.49

<0.001
4.
r
0.72
0.75
r
0.70
0.28
r
0.53
0.53
50.98 0.14 r 0.35
<0.001
3. 600.17 r 0.38
44.59 0.46 r 0.5044.32
37.3
35.29
<0.001
5.
r
0.49
0.69
r
0.46
0.36
r
0.48
0.40
28.86
28.37
<0.001
4. 400.28 r 0.53
r 0.720.75 r 0.70
0.5327.02
13.72 0.05 r 0.19
0.002
6.
0.19
r
0.39
0.03
r
0.19
<0.001
5. 200.36 r 0.48
0.69 r 0.46
0.40 r 0.49
Q
1.

0
The results are
expressed0.19
as Mr 0.39
r sd
0.002
0.03
0.19
The
resultsrare
expressed as0.05
M ± sdr 0.19
Year
1
Year
Year 3Q = question of
Fagerström
testof 2nicotine dependence(FTND)
Q = questionTheof Fagerström
test areof nicotine
dependence as(FTND)
results
expressed
M r sd

6.

0

14

-

0.001

5.39 r 2.09

Due toFig.
an increase
Fagerströmofscorein the yearof study, we examined
3. Theofpattern
nicotine
dependence
the evolution of subjects’severity
percentage distribution,
divided in categories of nicotine
on male groupdependence severity,
in malegppersons,as well pas =in female
subjects.
(p <both0.001,= 0.009,
0.002).
In Fig. 3 it is shown that there is a pattern in male subjects, characterized by ahigher percentage of high nicotine dependence subjects, which remains unchanged
along with the year of study (p = 0.86). This tendency was also revealed in caseof medium nicotine dependence (p = 0.42), and low nicotine dependence (p =0.74) percentage subjects, respectively. But, in case of female subjects (Fig.4), thepattern was modified along the year of study, in the way of a significant increase ofhigh nicotine dependence (p < 0.001), decrease of medium nicotine dependence(p = 0.007) percentage subjects, without any change of low nicotine dependence(p = 0.31) percentage subjects.
These results are also sustained by comparative analysis using severity categories of nicotine dependence in each gender, along with the year of study. Thus,in the first year of medical studies, male subjects were placed in the high nicotine
dependence category (X2 = 28.71, p < 0.001), while female subjects in low
nicotine dependence (X2 = 5.16, p < 0.05) and medium nicotine dependence (X2= 4.93, p < 0.05) category. In the second year, the male subjects remained in highnicotine dependence category (X2 = 4.01, p < 0.05), while in the third year there
were no significant differences related to gender, for any of the severity categories (X2= 3.50, p = 0.06 for low nicotine dependence ; X2 = 0.02, p = 0.96 for medium
nicotine dependence ; X2 = 2.07, p = 0.15 for high nicotine dependence).
Smoking habits of nicotine dependence
Different aspects of smoking behavior associated with nicotine dependence
were analyzed using FTND. Statistic analysis was based on comparing intermediate scores obtained at each of the 6 items (Q) of FTND, both in male subjects (Tab.
100

20

0.003

4.11 r 1.59

Medium40

p

Year 1



80

56.6

60

%

Q = question of Fagerström
of nicotine
Low test
MediumHighdependence (FTND)DISCUSSIONS

Resultsstudiesof
performed
in Romania
starting with the ’90
Fig.of4.population
The pattern
nicotine
dependence
revealed a concerning
evolutionofsmoking
prevalence,both within generalseverity on female groupg groups, such as female gender persons andpopulation, as well as within
population( p < 0.05, p < 0.001).
Fiziologia - Physiology  2009.19.2(62)adolescents, but also professional groups, such as doctors and pharmacists.
Dynamics of tobacco use in Romania between 1995 and 2000 indicates theincrease of adult smokers’ prevalence, from 28.5% in 1995 to 36.1% in 2000. This
increase is due mainly to the high prevalence of smoking in adult women, from
15.2% in 1995 to 25.0% in 2000, while the increase for the male subjects was from
42.7% in 1995 to 48.0% in 2000 (6).
Results of our study showed that smoking prevalence between the students
in the first three years of medical education was 34.28%, and 35.50% in malesubjects, and 33.03% in female subjects (p = 0.25). Our research also highlighted
the existence of an increasing tendency in new smoker’s percentage, but also innicotine dependence severity, along with year of study, both in male, as well as
in female subjects. However, prevalence of male smokers situated under the level
estimated for the entire population, while the female smokers’ prevalence washigher to the general population.
Compared with data from literature concerning smoking prevalence in students from medicine faculties in Europe, smoking prevalence in our subjects washigher than the same parameter evaluated in other countries such as Great Britain-11% (7), Czech Republic - 18% (8), Nederland - 18% (9), Hungary - 20.9% (10),or Germany - 23.7% (11), but was comparable with prevalence in students fromTurkey - 23.9% – 45.9% (12). A survey investigating the 4th year students from
Medical Faculty in Bucharest showed 32.7% smokers (30% females and 36.5%
males) and 7.8% former smokers (6).
Our study also revealed the elevated number of female medical students smoking
compared with the same population segment in the western countries (16% – 23%)(13), but which was very close to the same parameter in the eastern countries, such as
the former Yugoslavia (24% - 35.1%) (14) or Russia (30% – 35%) (15).
Our results suggest that male subjects became smokers previous to commitment to University and remained high degree of severity smokers at least in the first
three years of studies. On the other hand, female subjects became smokers only
after starting the medical education, and progressively acquired a smoking behavior
similar to male subjects. Following the same rationale, studies performed in Spain(16), Turkey (17) or Poland (18) showed that medical students are more likely to
become smokers after coming to University, rather than quit smoking, and seemto increase the number of smoked cigarettes, rather than decrease it.
High percentage of smokers revealed even from the first year of medical
education can be also analyzed from the perspective of smoking permanent increasing tendency among students in medium or high schools. Thus, numerous studies
conducted in Bucharest high schools showed that tobacco use generally starts at
the age of 14 (2.8%), significantly increase up to the age of 16 (12.3%), reachinga peak at the age of 18 (28.5%) (6). These studies also revealed the tendency ofpermanent increase in girls smoking during high school.
Although our study was limited to the first three years of medical educationand there were some inconveniencies related to the large number of female gendersubjects, considerably larger compared with the male subjects, the percentage increaseof new smokers, and also the reduced tendency of quitting smoking present in equal
proportion in both genders suggest the progressive development of smoking habit
even in the superior years of study. Evidence can consolidate our conclusion based on
results of enquiries performed between 1997 and 1999 among doctors in Romania,
which showed 43.2% prevalence of smoking doctors, with a pick of prevalence indoctors of 30 – 49 years old (44.3%). A high rate of smokers was also found in family
doctors (40% in males, and 19% in females), as well in specialist doctors (62.6%),
pneumologists (60.5% in males, and 40% in females) and surgeons (45.8%) (6).
Although some data from smoking habits among medical students study
are relatively old, the findings revealed that medical students generally havepoor knowledge of smoking related diseases as coronary artery disease, lung2009.19.2(62)  Fiziologia - Physiology

cancer, pulmonary emphysema and peripheral vascular disease, bladder cancer,
and neonatal mortality (19-21).The deficiencies in knowledge among medical
schools reflect a general failure of medical schools globally to include teaching
about tobacco in the curriculum.

CONCLUSIONS
The results of our study are far from being complete understanding of the subject.
Our finding can only suggest that the high rate and severity of smoking among medical
students may have an impact on public health, since medical students of today will bycalled upon to lead by either their non-smoking example or their clinical advice andpractice the way to the reduction of tobacco use and smoke-cessation.
The relatively less encouraging smoking data among our medical studentssuggest the need to promote tobacco education and intervention efforts within
this population segment.
REFERENCES
1. Collinshaw NE, Lopez AD. The tobacco epidemic: a global publichealth emergency. Tobacco Alert. Geneva. World Health Organization, 1996.
2. Wald NJ, Hackshaw AK. Cigarette smoking: an epidemiologicaloverwiew. Br Med Bull 1996; 52: 3 – 11.
3. Fiore MC, Epps RP, Manley MW. A missed opportunity. Teaching
medical students to help their patients successfully quit smoking.
JAMA 1994; 271: 624 – 626.
4. Heatherton TF, Kozlowski LT, Frecker R. The Fagerström test
for nicotine dependence: a revision of the Fagerström tolerance
questionnaire. Br J Addict 1991; 86: 1119 – 1127.
5. Patkar AA, Hill K, Batra V, Vergare MJ, Leone FT. A comparisonof Smoking Habits Among Medical and Nursing Students. CHEST
2003; 124: 1415 – 1420.
6. Vladescu C, Mihaltan F, Sanda L, Andrei C, Paunescu B, Paun C,
Pargioaga D, Loghin CR, Daramus I, Duta I. Cunostiinte, atitudini sipractici legate de consumul de tutun in randul populatiei generale
din Romania – CAPCTR – 2003. „Fumatul si sanatatea publica inRomania” 2003: 22 – 23.
7. Meakin RP, Lloyd MH. Disease prevention and health promotion: a study of medical students and teachers. Med Educ 1996;
30: 97 – 104.
8. Kralikova E, Kozak J, Rames J. Czech medical faculties and smoking. Centr Eur J Publ Health 1995; 3: 97 – 99.
9. Dekker HM, Looman CWN, Adriaanse HP. Prevalence of smoking
in physicians and medical students, and the generation effect in
the Netherlands. Soc Sci Med 1992; 36: 817- 822.
10. Pico B, Barabas K, Markos J. Health risk behavior of a medical
student population: report on a pilot study. J R Soc Health 1996;
116: 97 – 100.
11. Brenner H, Scharrer SB. Parental smoking and socio-demographic factors related to smoking among German medical
students. Centr Eur J Publ Helth 1995; 3: 97 – 99.
12. Kocabas A, Burgut R, Bozdemir M. Smoking patterns at different
medical school ?. Tobacco Contr 1994; 3: 228 – 235.
13. Crofton JW, Tessier JF, Freour PP. European medical schools andtobacco. Med Educ 1992; 7: 105-114.
14. Vlajinac H, Adanja B, Jarebisnki M. Smoking behaviour of
medical students in Belgrade (Yugoslavia). Eur J Epidemiol 1997;
7: 709-710.
15. Tessier JF, Freour PP, Mejiari C. Smoking behaviour and attitudes of medical students towards smoking and anti-smoking
campaigns in Austria, Japan, USA and the former URSS (Rusia andEstonia). Tobacco Control 1993; 2: 24 – 29.
16. Bonet RC, Granados CF, Vallescar I. Tobacoism in medical students. Atencion Primaria 1992; 9: 203 - 206.
17. Itil O, Ergor G, Ceylan E. Knowledge and Attitudes about smoking

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among students in a medical faculty. Turkish Resp J 2004; 5: 86 – 91.
18. Kozielsky J, Jastrebski D, Gabrys J. Changes of smoking habits
over 10 years among II years medical students. Pneumol Alerg
Polska 1996, 64, 50-53.
19. Crofton JW, Freour PP, Tessier TJ. Medical education on tobacco: implications of a worldwide survey. Med Educ 1994; 28:

187 – 196.
20. Crofton JW, Tessier TJ. A worldwide survey if knowledge andattitudes of tobacco medical students, In :”Educating medical
students about tobacco: planning and implementation”. Richmond R, Ed. Paris: International Union Against Tuberculosis andLung Disease, 1997.

TENDINTE IN STATUSUL SI OBICEIURILOR FUMATORILOR IN CADRULGRUPULUI DE STUDENTI LA MEDICINA DIN ANUL TREIREZUMAT
Scopul acestui studiu longitudinal a fost sa evalueze modificarea statusului si obiceiurilor fumatorilor in timpul primilor trei ani de studii medicale. In fiecarean, intre 2003-2006, au fost supravegheati 350 studenti la Medicina (126 baieti si 224 fete), folosind un chestionar care includea detalii demografice, datelegate de statusul de fumator si obiceiurile legate de fumat, care au fost evaluate prin testul Fagerström de dependenta la nicotina (FTND). Rezultatele studiului
nostru au aratat ca procentul subiectilor de sex masculin care erau fumatori curenti a fost semnificativ mai mare decat acelasi parametru evaluat la subiectii
de sex feminin doar in primul (p = 0,03) si al doilea an de studiu (p = 0,04). La subiectii de sex feminin, a fost semnificativa cresterea procentului de noi
fumatori relationata cu anul de studiu (p = 0,01). Severitatea dependentei de nicotina, estimata cu ajutorul scorului FTND, a crescut semnificativ in fiecare
an de studiu, atat la subiectii de sex masculin (p = 0,002), cat si la cei de sex feminin (p < 0,001). Este ingrijoratoare tendinta de consum a tutunului

in continua crestere mai ales intre subiectii de sex feminin. Datele relativ descurajatoare legate de fumat, in randul sudentilor la Medicina,sugereaza nevoia promovarii unei educatii anti-tabac si a unor campanii de interventie in randul acestui segment populational.
Cuvinte cheie: fumat, studenti medical students, chestionar Fagerström

16

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Fiziologia - Physiology  2009.19.2(62)ESTABLISHMENT, PROPAGATION AND MAINTENANCE
OF PRIMARY PULMONARY FIBROBLASTSBIANCA MATIS1, CARMEN BUNU2, GABRIELA TANASIE2, CALIN TATU1, FLORINA BOJIN2,
LUMINITA CERNESCU1, CARMEN TATU2, LAURA MARUSCIAC2, VIRGIL PAUNESCU2
University of Medicine and Pharmacy Victor Babes Timisoara
1
Department of Immunology and Transplant, County Emergency Clinical Hospital of Timisoara
2
Department of Physiology and Immunology

ABSTRACTPrimary fibroblasts represent a heterogeneous population of cells that can be separated into subsets based on cell markers. Obtaining fibroblasts culture
initially presume collecting tissue explants from tissues such as the lung, mechanically disrupted it and enzyme digested, in order to obtain an uni-cellularsuspension. Once established the primary culture of fibroblasts, it is necessary to characterize the new strain of cells to ensure their purity and fibroblastic
phenotype. The methods described in this paper outline the procedure used to initiate and establish primary fibroblast from pulmonary tissue and fibroblast
characterization using morphological features and fibroblast markers, like vimentin, alpha-smooth muscle actin (α-SMA) and myosin.
Key words: fibroblasts, cell culture, morphologic features, immunohistochemistry

INTRODUCTION
Asthma is a chronic inflammatory disease of the airway characterized by
airflow limitation and airway hyperresponsiveness (AHR) to nonspecific irritants.
The AHR is defined by exaggerated airway narrowing, which can usually be reversedby bronchodilators that relax airway smooth muscle (ASM). The observations thatthe basic features of asthma are associated with airway inflammation and changes
in airway structure have led to the predominant view in the past decade that ASM
was primarily an effecter, whereas airway inflammation was thought to be thecausal pathophysiological mechanism underlying AHR (1). This concept has beenstudied, showing dissociations between AHR and airway inflammation. The chronic
inflammatory response in the airway in asthma is characterized by the presenceof increased numbers of Th2 lymphocytes, eosinophils, and activated mast cells(2). Besides the presence of inflammatory cells in the airway, there are different
levels of structural changes termed airway remodeling (3). Characteristic structuralchanges of airway remodeling include epithelial cell mucus metaplasia, smoothmuscle hypertrophy/hyperplasia, subepithelial fibrosis, and increased angiogenesis.
Increased myocyte smooth muscle mass, is thought to be due to an increase in myofibroblast proliferation (4) and a more specific role in this has the relation between
fibroblasts and myofibroblasts (5). Fibroblasts are a population of heterogeneous
cells which play a vital role in maintaining the structural integrity of organs suchas lungs, skin, heart, sclera, and the orbit, in both physiological and pathologicalcircumstances, like healing and reparative processes, and in the pathogenesis ofscarring (fibrosis) disorders. Fibrosis is the result of fibroblast hyperplasia, as well
as abnormal and accelerated production of extracellular matrix. These alterations
are succeeded by fibroblast activation (6). Even if the fibroblasts don’t normally
have contractile elements, they are able to develop a minimal contraction, due
to cytoskeleton rearrangement, enabling them to migrate to affected areas andto secrete matrix proteins, this meaning in fact the fibroblasts-myofibroblasts
transformation (7). That myofibroblasts represent an intermediate state between
fibroblasts and smooth muscle cells is best demonstrated in the prostate (8), in the
pericytes surrounding the fetal vessels of the placental stem villi (9) and in stromal

myofibroblasts of the breast (10). In both cell culture in vitro and native tissues in
situ, myofibroblasts possess several distinguishing morphological characteristics.
They display prominent cytoplasmic actin microfilaments (stress fibers), and they areconnected to each other by adherence and gap junctions (11). Immunohistochemical characterization of myofibroblasts is based on antibody reactions for two of thethree filament systems of eukaryotic cells (12). These three systems are composedof actin, a component of the microfilaments, vimentin, desmin or lamin, membersof the intermediate filament system and the tubulins of the microtubules. Myofibroblasts have not been characterized with regard to tubulins. Beta and gamma
actins are expressed by all cells, including myofibroblasts, which may also express
α-SMA. Myofibroblasts stain negatively for alpha-cardiac and alpha-skeletal actin(13). Myofibroblasts are not well characterized regarding myosin isoforms (12).
Vimentin, desmin, and α-SMA are the three filaments most often used to classify
myofibroblasts (14). Expression of these proteins may vary with the tissue studied,within species and whether the cells are studied in situ or in culture and, even in agiven tissue, if the cells are activated by hormonal or cytokine treatment or by disease(15). Based on immunohistochemical staining of these filaments, a classification
system has been proposed (9). Myofibroblasts that express only vimentin are termed
V-type myofibroblasts, those that express vimentin and desmin are called VD-type,those that express vimentin, α-SMA, and desmin are called VAD-type, those that
express vimentin and α-SMA are called VA-type, and those that express vimentin
and myosin are called VM-type.

MATERIAL AND METHODS•

Pulmonary explants collection

The samples were collected during pulmonary lobectomy, from patients withlung cancer. The samples were obtained from 5 patients (4 male, 1 female), aged
54.6±16.8, from areas distal the tumor, which were histological free of cancer. All

Received March 2009. Accepted May 2009. Address for correspondence: Dr. Bianca Matis, Physiology and Immunology Department, “Victor Babes”University of Medicine and Pharmacy Timosoara,
2A Eftimie Murgu Square, 300041, Timisoara, phone: 0724217076, e-mail: bianca.matis@yahoo.com
2009.19.2(62)  Fiziologia - Physiology

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samples were collected after the patients had signed the informed consent. The
samples were collected in 50 ml Falcon tubes, in PBS supplemented with Penicillin
200 UI/ml, Streptomycin 150 μg/ml and Gentamicin 50 μg/ml. The samples werestored at 4°C until processing.
•

Isolation and cultivation of primary fibroblasts

The samples were processed in 100 mm2 Petri dishes. The bronchial andvascular structures were removed from the sample, and afterwards the lung parenchyma was cut in <0.5 cm pieces with a sterile scalpel. The samples were thenwashed twice with PBS at room temperature, to remove as many erythrocytes as
possible. The tissue was then processed in three different ways in order to obtaincells from lung parenchyma, using aseptic techniques, for minimal contaminationof the primary culture.
a. Lung explants: small pieces (less than 0.5 cm across) are placed in 25 mm2Petri dishes which were previously held for 30 minutes at 37°C, in order to increase
surface adherence (Fig. 1). Then the lid is placed on top of the plate, for 10 minutes,
at room temperature, in order for the tissue to adhere to the plate. Afterwards, thetissue is resuspended in DMEM High Glucose culture medium supplemented withFCS 20%, L-Glutamine 2 mM, Penicillin 150 UI/ml, Streptomycin 100 μg/ml. The
plates are then stored for 4 days without changing the medium. After the 4 days,
the medium is changed twice a week, until a confluence of 60-70% is reached.

Fig. 1. Pulmonary explants

Fig. 1. Pulmonary explants
Fig. 1. Pulmonary explants

b. Collagenase digestion: the lung parenchyma fragments obtained after
removal of connective bronchial and vascular tissue are placed in a 15 ml Falcon
tube with 5 ml collagenase 200 UI/ml, for 30 minutes at 37°C. In order to obtaina uni-cellular suspension, the tissue is filtered through a 70 μm pores cell strainer,
washing continuously with PBS. The cell suspension thus obtained, together withthe collagenase solution in which the tissue was kept, are centrifuged at 1500 rpm
for 8 minutes, at room temperature. The supernatant is removed with a Pasteurtransfer pipette, and the cell pellet is resuspended in 7.5 ml DMEM High Glucose
culture medium supplemented with FCS 20%, L-Glutamine 2 mM, Penicillin 150
UI/ml, Streptomycin 100 μg/ml, and placed in a T25 culture flask with filtered cap.
The flask is incubated at 37°C, a relative humidity of 95%, and the level of CO2 ismaintained at 5%. The medium is changed after 4 days.
c. Trypsin digestion: the lung parenchyma from the biopsies is placed in a 15 ml
Falcon tube, in 5 ml trypsin 0.25%, for 30 minutes at 37°C. For obtaining the unicellular suspension, the tissue is filtered through 70 μm pores cell strainer, washingcontinuously with PBS. The cell suspension plus the trypsin solution, in which thetissue was kept, are centrifuged at 1500 rpm for 8 minutes, at room temperature.
The supernatant is removed with a Pasteur transfer pipette, and the cell pellet isresuspended in 7.5 ml DMEM High Glucose culture medium supplemented with FCS
20%, L-Glutamine 2 mM, Penicillin 150 UI/ml, Streptomycin 100 μg/ml, and placedin a T25 culture flask with filtered cap. The flask is incubated in same conditions as
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previously described. The medium is changed after 4 days.
During the first two weeks, the cells which are not fibroblasts do not adhere to
the plate surface and are weeded out, while the fibroblasts proliferate, becoming thedominant cells in the culture. Once the fibroblasts from the explants have grown,or the ones from the cell suspension have adhered, forming a cell monolayer witha confluence of at least 80%, the first cell passage is possible.
Cell passaging is made by trypsinization, following the next steps:- The cells are washed once with PBS;
- They are detached from the plate surface by adding 5 ml of 0.25% trypsin
EDTA solution, at room temperature (the trypsin-EDTA solution should not be left
on the cells for longer then 5 min.);- Once the cells have separated from the plate, they are resuspended in an equalvolume of 4°C complete cell culture media for neutralizing the trypsin action;- The obtained cell suspension is placed in a 15 ml Falcon tube and centrifuged
at 1500 rpm for 5 minutes, at room temperature;- The cell pellet is resuspended in 1 ml culture medium;- The cells are then counted with a hemacytometer, according to standard
cell culture methods;- The cells are then seeded in T25 or T75 flasks containing cell culture media(depending on the number of cells), for a density of 1x104 cells/cm2.
- The growth medium is changed twice a week until the next passage;- The primary fibroblasts can be used between 3rd and 10th passage, and they
can be stored indefinitely in liquid nitrogen.
Freezing the cells:- The cells are rinsed once with PBS;
- They are detached from the plate surface by adding 5 ml of 0.25% trypsin
EDTA solution, at room temperature;- The trypsin action is attenuated by adding 5 ml of culture medium supplemented with FCS 20%, Penicillin 1%, Streptomycin 1%;- The cell suspension is placed in a 15 ml Falcon tube and centrifuged at 1500
rpm for 5 minutes, at room temperature;- The cell pellet is resuspended in 1 ml FCS;
- The cells are then counted with the hemocytometer;- The freezing solution is prepared, using FCS + 20% dimethylsulfoxide(DMSO);- The cells are placed in 2 ml cryotubes, 1.5x106 cells/cryotube, using in 1:1
cell suspension and freezing solution.
• Confirmation of fibroblasts culture
by immunohistochemistry
The cells obtained from lung biopsies were analyzed immunohistochemically.
The cell characteristics involve surface or intracellular markers, as well as the cell
fenotype. The fibroblasts have a typical morphology. They are elongated cells andhave an oval nucleus. They can be star-like, with irregular cytoplasmic prolongations.
Lung fibroblasts express Vimentin and Collagen, while CD45, cytokeratin – epithelial
cell marker, and smooth muscle actin – expressed by myofibroblasts and smoothmuscle cells are consistently absent (6).
For the immunohistochemical analysis, the cells were placed in 4 well plates, at
4000 cells/cm2 cellulr density, and 7000 cells/well. After 4 days of incubation at 37°C,
when the cells have reached a confluence of 60-70%, the medium was removed,
the cells washed twice with PBS, and immunohistochemistry was performed forVimentin, actin and myosin, following the next steps:
1. The cells are fixed by washing with 0.02 % formaline, for 5 minutes.
Fiziologia - Physiology  2009.19.2(62)2. Rinse gently with PBS at room temperature, for 5 minutes.
3. To stain the cells for Vimentin, actine, or myosine incubate cultures withthe primary antibody anti-vimentin (Dako, N1521), anti- α-SMA clone 1A4(Dako, N1584), or anti- havy chain myosine (Dako, M3558), diluted in PBS, atroom temperature, for 10 minutes. These reagents are supplied ready-to-use, so
blocking is unnecessary.
4. The cells are washed twice with PBS, for 2 minutes, at room temperature.
5. The secondary biotinylated antibody is added, for 10 minutes, at room
temperature.
6. The cells are washed twice with PBS, for 2 minutes, at room temperature.
7. The Streptavidin-HRP is a applied enough to cover specimen, for 10 minutes,
at room temperature.
8. The cells are washed twice with PBS, for 2 minutes, at room temperature.
9. The substrate (AEC) is added, 2 drops/well, for 10 minutes, until the color
turns red.
10. The cells are washed with distilled water, for 5 minutes.
11. Hematoxylin is added, for 30 seconds.
12. The cells are washed with distilled water, for 10 minutes.
13. The mounting solution and the cover slip are applied.

ogy, and concerning the markers, they respected the fibroblasts phenotype. All cells
stained positive for Vimentin and negative for α-SMA and myosine (Fig. 4).

• In vitro fibroblasts differentiation through myofibroblasts
The following experiment will be designed to assay the effect of pro-inflammatory factors on the phenotypic change of fibroblasts. The cells will be plated in
6 well plates (4000 cells/cm2), referring as day 2. Twenty-four hours later (day 1)
the FCS concentration will be reduced to 4% to decrease the proliferating rate andmaintained for the rest of the experiment. Twenty-four hours later the medium
will be replaced by medium containing IL-4 (R&D, 204-IL-050, 25 ng/ml), IL-13(R&D, 213-IL-025, 25 ng/ml), TGF- β1 (Sigma Aldrich, T7039, 1.5 ng/ml), TGF- β2(Sigma Aldrich, T2815, 1.5 ng/ml) and one medium which will contain a mixtureof all factors in same concentrations as above. One well will be maintained withdiluents only (0 ng/ml factor). This will be referred as day 0. Each experiment will
be proceeded in duplicate – one sample for reverse transcription polymerase chain
reaction (RT-PCR) and the other for immunohistochemistry. The cells pulsed for 14days, will be assessed for Vimentin, alpha-smooth muscle actin (α-SMA), myosine
havy chain and desmine.

A

RESULTS• Isolation and cultivation of primary fibroblasts
The human primary lung fibroblasts were isolated by using all of the threemethods presented above (lung explants, collagenase digestion and trypsin digestion),but the establishment of cell cultures and the cell passaging were obtained only byusing the digestion methods. The cells obtained from the lung explants resembled
fibroblasts morphologically (Fig. 2), but after two weeks of cultivation they did notreach a confluence of at least 70%, and detached themselves from the plate surface.
Lung fibroblast cultures were obtained by using both digestion methods, butwith collagenase, were obtained more uniform cultures (Fig. 3).
• Confirmation of fibroblasts culture byimmunohistochemistry
The cells obtained from pulmonary biopsies presented the fibroblasts morphol2009.19.2(62)  Fiziologia - Physiology

Fig. 2. Pulmonary fibroblasts isolated from tissue explants - 14 days culture
Fig. 2. Pulmonary
fibroblasts
fromtissue
tissueexplants- 14culturedays culture
Fig. 2. Pulmonary
fibroblastsisolated
isolated fromexplants- 14 days

A
A

B
B

Fig. 3. Pulmonary
fibroblasts
isolatedby colagenase(A) colagenase digestion andtrypsindigestion
Fig. 3. Pulmonary
fibroblasts
isolatedby (A)
and(B)
trypsindigestion
Fig. 3. Pulmonary
fibroblasts
isolatedby (A) colagenasedigestion
digestion and(B)(B)
trypsindigestion

A

B

B

C

C

Fig.4. Representativestaining
immunohistochemicalof positive- vimentin) and negative (B - Į-SMA an
Fig. 4. Representative immunohistochemicalof positive (Astaining- vimentin)and(Anegative
fibroblasts markers(B - α-SMA and C - myosine) fibroblasts markers

Fig.4. Representative immunohistochemical staining of positive (A - vimentin) and negative (B - Į-SMA and C - myosine)
fibroblasts markers

CONCLUSIONS AND DISCUSSIONS
1. Primary fibroblast cultures may be initiated from lung tissue, but not limited
to it and could be extended at various organs.
2. Mechanically disrupting tissue can be used for isolate fibroblasts, but alternative methods including enzymatic dissociation with collagenase are preferable inorder to obtain primary fibroblasts strains.
3. Once established the fibroblasts cultures, media should be replaced twicea week and the cultured cells used between passages 3 and 10, because primary
fibroblast strains have a finite life span.
4. Fibroblasts at an early passage may be frozen and stored indefinitely in liquid
nitrogen. For limited period of time cells can be kept frozen in isopropanol freezing
containers at –80°C. Before freezing, cells should be counted and resuspended infreezing media (FCS supplemented with 10% DMSO).
5. Cultures derived from lung tissue usually consist of fibroblasts only, becauseof the selecting media culture, but it is important to document the purity of eachstrain. The usual strategy involves assessing the presence of cell markers andphenotypic proprieties of fibroblasts.
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Acknowledgements: The study was supported by CNMP Research
Grant PREVALERG no. 41-011/2007.
REFERENCES
1. An SS, Bai TR, Bates JHT, Black JL, Brown RH, Brusasco V, et al.
Airway smooth muscle dynamics: a common pathway of airway
obstruction in asthma. Eur Resp J 2007; 29:834-860.
2. Cohn L, Elias JA, Chupp GL. Asthma: Mechanisms of Disease Persistence and Progression. Annu. Rev. Immunol 2004;22:789-815.
3. Boulet LP, Sterk PJ. Airway remodeling: the future. Eur Resp J
2007; 30:831-834.
4. Lloyd CM, Robinson DS. Allergen induced airway remodeling.
Eur Resp J 2007;29:1020-1032.
5. Pascual RM, Peters SP. Airway remodeling contributes to theprogressive loss of lung function in asthma: an overview. J Allergy
Clin Immunol 2005:477-486.
6. Baglole CJ, Reddy SY, Pollock SJ, Feldon SE, Sime PJ, Smith
TJ, Phipps RP. Isolation and phenotypic characterization oflung fibroblasts. Methods in Molecular Medicine 2005;117:115127.
7. Singh SR, Hall IP. Airway Myofibroblasts and Their Relationship with Airway Myocytes and Fibroblasts. Proc Am Thorac Soc
2008;5:127–132.

8. Kassen AD, Sutkowski M, Ahn H, Sensibar JA, Kozlowski JM,
Lee C. Stromal cells of the human prostate: initial isolation andcharacterization. Prostate 1996;28:89-97.
9. Kohnen G, Kertschanska S, Demir R, Kaufmann P. Placental villous stroma as a model system for myofibroblast differentiation.
Histochem. Cell Biol. 1996;105:415-429.
10. Ronnov-Jessen L, Petersen OW, Koteliansky VE, Bissell MJ. The
origin of the myofibroblasts in breast cancer: recapitulation oftumor environment in culture unravels diversity and implicatesconverted fibroblasts and recruited smooth muscle cells. J. Clin.
Invest. 1995;95:859-873.
11. Darby I, Skalli O, Gabbiani G. Alpha-smooth muscle actin istransiently expressed by myofibroblasts during experimental
wound healing. Lab. Invest. 1990;63:21-29.
12. Fuchs E and Cleveland DW. A structural scaffolding of intermediate filaments in health and disease. Science 1998;279:514-519.
13. Schmitt-Graff A, Desmouliere A, Gabbiani G. Heterogeneityof myofibroblast phenotypic features: an example of fibroblastic
cell plasticity. Virchows Arch. 1994;425:3-24.
14. Mermall V, Post PL, Mooseker MS. Unconventional myosins incell movement, membrane traffic, and signal transduction. Science
1998;279:527-533.
15. Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West
AB. Myofibroblasts. II. Intestinal subepithelial myofibroblasts. Am.
J. Physiol. 277 (Cell Physiol 46).

STABILIREA, PROPAGAREA SI MENTINEREA CULTURII PRIMAREFIBROBLASTICEREZUMAT

Fibroblastii primari reprezinta o populatie celulara heterogena, ce poate fi impartita in subtipuri celulare, pe baza marker-ilor caracteristici. Obtinerea unei
culturi celulare presupune initial prelevarea tesutului pulmonar, segmentarea sa si digestia enzimatica pentru a forma o suspensie uni-celulara. Dupa stabilirea culturii de fibroblasti este necesara caracterizarea celulelor derivate din prima generatie de celule, pentru a stabili omogenitatea poulatiei. Metodele
descrise in continuare releveaza procedura de obtinere si perpetuare a unei culturi de fibroblasti din tesut pulmonar, precum si caracterizarea celulelor folosind
caracteristicile morfologice si markeri imunohistochimici, precum vimentina, alfa-actina de muschi neted si miozina.
Cuvinte cheie: fibroblasti, culturi celulare, caracteristici morfologice, imunohistochimie

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Fiziologia - Physiology  2009.19.2(62)DENDRITIC CELLS AND T CELLS ACTIVATIONLUMINITA CERNESCU1, JANINA JIGA2, LUCIAN JIGA2, CARMEN BUNU1, CALIN CERNESCU3,
BIANCA MATIS1, FLORINA BOJIN1, CARMEN TATU1, GABRIELA TANASIE1, VIRGIL PAUNESCU1
University of Medicine and Pharmacy Victor Babes Timisoara, Romania
1
Physiology and Immunology Department
2
Pius Branzeu Center for Laparoscopic Surgery and Microsurgery
3
Department of Gastroenterology and Hepatology

ABSTRACT
Dendritic cells are the most important antigen presenting cells that possess the unique ability to stimulate naïve T cells. DCs are derived frombone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. They are enables to havedirect contact with incoming antigens, take up, process and present them on the cell surface linked to major histocompatibility molecules.
After antigen uptake, DCs undergo further maturation and migrate to draining lymph nodes, where they present antigens to T cells and induce
an immune response toward a Th1 or a Th2 profile.
Key words: dendritic cells, costimulatory molecules, T cells, Th1/Th2 response, cytokine

ANTIGEN-PRESENTING CELLS – GENERALCONSIDERATIONS
Immune response initiation and establishment are based on a close interactions between antigen-presenting cells (APCs) and T lymphocytes. It is known
that T lymphocytes cannot recognize antigens in their native form. They require
antigen-presenting cells for processing antigens into short fragments and presenting them on major histocompatibility complex molecules (MHC). APCs include
macrophages, B lymphocytes and dendritic cells (DCs). These specialized cells are
distinguished by two important properties: class II MHC molecules expression on
their membranes and the ability to deliver a co-stimulatory signal necessary for
T cell activation. DCs are the most effective antigen-presenting cells, expressing
high levels of class I and class II MHC molecules, as well as high levels of B7-1 andB7-2 glycoproteins, the principal co-stimulatory molecules. For this reason, DCs arevery potent activators of naive, memory and effector T cells. In contrast, activatedmacrophages are common activators of memory and effector T cells, but are notable to activate naive T cells. This type of APCs up-regulate their expression of class
II MHC molecules and co-stimulatory B7 molecules. Resting B cells express class II
MHC molecules, but fail to express co-stimulatory B7 molecules and cannot activate
naive T cells. Activated B cells up-regulate their class II MHC molecules expression
and begin expressing B7 molecules, being able to activate now naive as well as
the memory and effector T cells (1).

DENDRITIC CELLS
DCs were described in all lymphoid organs, blood, bone marrow, and in several
other organs, including the lung, liver, heart, kidney and urogenital tract (2). The

isolation of these cells from tissue samples is difficult because the conventional
procedures for cell isolation damage their long extensions, but are developed isolation techniques including enzymatic organ digestion, gradient centrifugations andgentler dispersion (3). In contrast to other leukocytes, DCs cannot be immunophenotypically identified using one single antigenit marker. They are usually identified
by the very intense expression of class II MHC molecules (HLA-DR) and the absenceof T, B, NK cell, monocytic and granulocytic lineage markers.
The major histocompatibility complex function as antigen-recognition
molecules, but they do not possess the fine specificity for antigen characteristicof antibodies and T cell receptors. The MHC loci encode two major classes ofmembrane-bound glycoproteins: class I and class II MHC molecules. Generally,
T helper cells (Th) recognize antigen combined with class II MHC molecules,
whereas cytotoxic T cells (Tc) recognize antigen combined with class I MHC
molecules (4).
Although most mature dendritic cells have the same function, the presentationof antigen to T cells, there are known four types of dendritic cells: myeloid dendritic
cells (mDCs), limphoid dendritic cells (pDCs), Langerhans cells and interstitialdendritic cells. They descend from hematopoietic stem cells through both the
myeloid and lymphoid lineages. The most relevant distinction between mDCs andpDCs is functional because these cells are activated by a different set of pathogenic
stimuls (5).
DCs are strategically mobilized to anatomic sites with high antigen exposure,thanks in part to their sensitivity to a whole array of chemoattracting inflammatory signals.
Because of theirs location, immediately above the basement membrane of
the epithelium, DCs are enables to have direct contact with incoming antigens(6). Mucosal DCs, named immature DCs, have a high endocytic and processing

Received March 2009. Accepted May 2009. Address for correspondence: Dr. Luminita Cernescu, Physiology and Immunology Department, “Victor Babes” University of Medicine and Pharmacy
Timosoara, 2A Eftimie Murgu Square, 300041, Timisoara, phone: 0766264850, e-mail: luminitabuda@yahoo.com
2009.19.2(62)  Fiziologia - Physiology

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Table I. Characteristics
of immature dendritic
cells cells
Table I. Characteristics
of immaturedendritic

Very intense expression of CD1a and CD11c
Low expression of HLA-DR
High endocytic and processing the antigens activity
Weak capability to activate T cells
Maturation inhibited by IL-10
Table II. Characteristics
mature dendritic
cells cells
Table II. Characteristics
ofofmaturedendritic

Very intense expression of HLA_DR, CD80 and CD86
Intense expression of CD83 and CD1a
Low expression of CD14
Weak capacity to take up antigens
Ability to stimulate naive T cells
Intense IL-10 production

the antigens activity and a weak capability to activate T cells. After antigen
uptake, they lose their capacity to take up antigens, become mature DCs, andcarry processed antigen from nonlymphoid tissues to the draining lymph node,
where they become able to stimulate naive T cells and induce the polarization of
the immune response toward a Th1 or a Th2 profile (7,8).Table I. Characteristics
of immature dendritic cells
There is another type of dendritic cells, named follicular dendritic cells,
which do not function as APCs. These cells do not express class II MHC molecules,
but express high levels of antibody membrane receptors. They are located inlymph node follicles, which are rich in B cells, playing an important role in B cells
response (9).
Antigens are not generally recognized in their entirety by lymphocytes.
Exogenous antigens, produced outside of the cells, are internalized by APCs, either
by phagocytosis or by endocytosis, degraded into peptide fragments and thendisplayed on their surface bound to a class II MHC molecule. Exogenous peptide
presentation is restricted to APCs, because class II MHC molecules expression islimited to these cells. CD4+ T cells recognize antigens combined with this moleculeclass. Endogenous antigens, produced within the host cells itself, are degraded into
peptide fragments that bind to class I MHC molecules within the endoplasmic
reticulum, from where are then transported to the cell membrane. Class I MHC
molecules are present on all nucleated cells and antigens combined with thesemolecules are recognized by CD8+ T cells (10).

T CELLS SUBSETS
Expression of CD4 or CD8 defines two major types of T lymphocytes. CD4+
T cells generally function as T helper lymphocytes because they help phagocytes
to destroy ingested microbes and stimulate B lymphocytes to produce antibodies.
This type of T lymphocytes is class II MHC molecules restricted. CD8+ T cells arecalled cytotoxic T lymphocytes because they kill other cells which contain intracellular microbes, and are class I MHC molecules restricted. There are another classof lymphocytes, called natural killer (NK), because they function as mediators ofinnate immunity (11).

IMMUNE RESPONSE ORIENTATION

ognized and responsed to an antigen. Th lymphocytes are activated by recognitionof antigen-class II MHC complex on APCs. During the primary immune response are
generated memory lymphocytes. Subsequent encounters with the same antigen
generate clone of memory cells and activates previosly generated cells, leading toa better immune response, called secondary immune response. These lymphocytes
secrete diferent cytokines, which play an essential role in the activation of other cells
participating to the immune response. Pattern nature of these cytokines influence
the orientation of immune response toward a Th1 or Th2 profile. Th1 cells are theinflammatory reaction support promoting cellular immune response through Tlymphocytes and macrophages activation. In contrast, Th2 cells are involved in the
humoral response and allergic reaction.
Differentiation into Th1 or Th2 response depends on many factors like: thenature and the dose of the antigen, the route of exposure, the genetic backgroundof the individual, type of costimulatory molecules expressed on the surface of theDCs and the polarizing cytokines microenvironment during antigen presentation.
It is known that the type of costimulatory molecules expressed on DCs isessential for determining Th differentiation. Mature DC is characterized by a very
intense expression of HLA-DR, CD80 and CD86 (HLA-DR+++/CD80-CD86+++), an
intense expression of CD83 and CD1a (CD83++/CD1a++) and a low expression of
CD14 (CD14-). The most important DCs costimulatory molecules involved in Th
differentiation are CD80 and CD86. The role of CD86 molecule seems to be more
important than CD80 in the induction of a Th2 response. In contrast, CD80 ispreferentially associated with Th1 type T-cell responses (12). It was observed that
B cells from patients with atopic dermatitis express CD86, and this expression iscorrelated with the total serum IgE level (13).
The orientation toward a Th1 or a Th2 profile is also dependent on the polarizing cytokines present during the interaction of DCs with T cells, IL-10 and IL-12,
both produced by DCs. It is postulated that IL-10 inhibits type 1 response, thus
promoting a Th2 response. In contrast, IL-12 favours Th1 responses enhancing IFN-γ
production by T cells (14). This cytokine stimulates, also, TNF-α production andreduces IFN-γ suppression IL-4 mediated (15). On the other hand, IFN-γ and evenIL-4 enhance IL-12 production induced by appropriate stimuli, while prostaglandin
E2 and IL-10 have an inhibitory effect (16). Levels of IL-12 in DCs vary geneticallyand with age, low levels promoting a Th2 response (17). IFN-γ secretion by Th1
cells activates macrophages, increasing their microbicidal activity and induces IgG
antibody-class that supports phagocytosis and complement fixation. In addition,
IFN-γ and IL-2, produced by the same cells, promote the Tc cells differentiationfrom CD8+ precursors and inhibits the Th2 cells expansion. Th2 cells secrete IL-4
and IL-5, which induce humoral immune responses caracterized by tendency to
produce excessive IgE such as in allergic immune reaction. There is a close association
between high IL-4 production and atopy. IL-4 seems to play a role in prevention of
autoimmunity by down-regulation of Th1 cells. The tissue affected by organ-specificautoimmune diseases present low levels of IL-4 production (18). It seems that thecapacity to induce Th2 immune response is a property of DCs which do not produce
Th1 polarizing cytokines, because they have losed their IL-12 or IFN-γ producing
capacity under some conditions16. Some authors report that the dynamics of DC
migration to the draining lymph nodes may result in rapid changes in DC composition and cytokine content in T cell areas. Because of that, during the early phases of
the immune response, Th1 cells are preferential primed, and this may be followedby priming of Th2 cells, in the later phases. Explanations of this could be that, inthe early phases of immune response, stimulated DCs enter in T cell areas in large
numbers and in the later phases, the influx of DCs decreases and the surviving DCs
lose their capacity to produce IL-12 (19).

The first exposure to antigen generate an immune response, called primary
immune response, mediated by naive lymphocytes, which not had previously rec22

CMYK

Fiziologia - Physiology  2009.19.2(62)CONCLUSIONS AND FUTURE ADVANCES
All these significant observations suggest that dendritic cells and otherantigen-presenting cells have main importance in immunity. They play two essential roles in immune response mediation : naive Th cells activation and activatedTh cells polarization toward Th1 or Th2 effector cells. Th cells differentiation may
depend on both the type of APCs and microenvironment signals. Because of theirfundamental role in initiating T cell responses, they could have the same relevancein many aspects of immune regulation, including autoimmunity and cancer, due
to their role in anti-cancer host responses and potential use as biological adjuvantsin tumour vaccines. In this case it becomes more clearly that the new strategies fordifferent diseases therapies should be focused them because they are a powerfultool for manipulating the immune system.
REFERENCES
1. Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen processing machines. Cell 2001 Aug 10; 106(3):255-8.
2. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ,
Pulendran B, Palucka K. Immunobiology of dendritic cells. Annu
Rev Immunol 2000;18:767-811.
3. Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell
polarization. Nat. Rev. Immunol. 2003;3:984-993.
4. Santambrogio L, Strominger JL. The Ins and Outs of MHC ClassII Proteins in Dendritic Cells. Immunity 2006; 25:857-859.
5. Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA, Bazan
F, Liu YJ. Subsets of human dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens.
J Exp Med 2001;194:863-869.
6. Jahnsen FL, Moloney ED, Hogan T, Upham JW, Burke CM, Holt
PG. Rapid dendritic cell recruitment to the bronchial mucosa ofpatients with atopic asthma in response to local allergen challenge.
Thorax 2001;56:823-6.
7. Huh JC, Strickland DH, Jansen FL et al. Bidirectional interactions
between antigen-bearing respiratory tract dendritic cells (DCs) andT cells precede the late phase reaction in experimental asthma:

DC activation occurs in the airway mucosa but not in the lungparenchyma. J Exp Med 2003;198:19-30.
8. Lambrecht BN, De Veerman M, Coyle AJ, Gutierrez-Ramos JC,
Thielemans K, Pauwels RA. Myeloid dendritic cells induce Th2
responses to inhaledantigen, leading to eosinophilic airway
inflammation. J Clin Invest.2000; 106:551-559.
9. Van Nierop K, De Groot C. Human follicular dendritic cells:
function, origin and development. Semin Immunol. 2002
Aug;14(4):251-7.
10. Castellino F et al. Chemokines enhance immunity by guiding
naive CD8+ T cells to sites of CD4+ T cell–dendritic cell interaction.
Nature 2006;440:890-895.
11. Maloy KJ, Powrie F. Regulatory T cells in the control of immune
pathology. Nature Immunol.2001;2:816-822.
12. Kuchroo VK, Das MP, Brown JA, et al. B7–1 and B7–2 costimulatory molecules activate differentially the Th1/Th2 developmental
pathways: application to autoimmune disease therapy. Cell.
1995;80:707-718.
13. Jirapongsananuruk O, Hofer MF, Trumble AE, Norris DA, Leung
DY. Enhanced expression of B7.2 (CD86) in patients with atopic
dermatitis: a potential role in the modulation of IgE synthesis. J
Immunol. 1998;160:4622-27.
14. Lore K, Sonnerborg A, Spetz AL, Andersson U. Immunocytochemical detection of cytokines and chemokines in Langerhans
cells and in vitro derived dendritic cells. J Immunol Methods.
1998;214:97-111.
15. Mosca PJ, Hobeika AC, Clay TM, Nair SK, Thomas EK, Morse MA,
Lyerly HK. A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40
ligand and interferon-γ treatment. Blood 2000;96:3499-3504.
16. Lanzavecchia A, and Sallusto F. Regulation of T cell immunity
by dendritic cells. Cell. 2001;106:263-266.
17. Upham JW, Lee PT, Holt BJ et al. Development of interleukin-12-producing capacity throughout childhood. Infect Immun 2002;70:6583-8.
18. O'Garra A, Steinman L, Gijbels K. CD4+ T cell subsets in autoimmunity. Curr Opin Immunol. 1997;9:872-83.
19. Lanzavecchia A, Sallusto F. Dynamics of T Lymphocyte Responses: Intermediates, Effectors, and Memory Cells. Science
2000;290:92-97.

CELULELE DENDRITICE SI ACTIVAREA LIMFOCITELOR T
REZUMAT

Celulele dendritice sunt cele mai importante celule prezentatoare de antigen caracterizate prin capacitatea unica de a stimula limfocite T naïve. Aceste celule
provin de la nivelul maduvei hematogene si circula in sange sub forma de celule dendritice imature. In tesuturile periferice ele vin in contact cu antigenele, pecare le capteaza, le proceseaza si le prezinta la suprafata celulei pe complexului major de histocompatibilitate. Odata cu preluarea antigenului ele devin celuledendritice mature si migreaza in ganglionii limfatici, unde stimuleaza limfocitului T inducand diferentierea raspunsului imun spre unul de tip Th1 sau Th2.
Cuvinte cheie: celule dendritice, molecule co-stimularoare, cellule T, raspuns Th1/Th2, citokine

2009.19.2(62)  Fiziologia - Physiology

23

CMYK

CORRELATION OF ANTHROPOMETRIC PARAMETERS WITH
AGE AND SEX IN POST PUBERTYBAGIU RADU1, ROTARU IULIA2“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
2
Emergency Public Hospital Timisoara

1

ABSTRACT
Postpuberty, with limits between 15-19 years old, is dominated by the reduction in growth velocity of height and weight, with the installation of puberty. It
was studied a representative population of 2908 adolescents from Timis county of 15-19 years old. Anthropometry was the work method. The results showeda continuous increase of height and weight in both genders; higher mean values in boys than in girls for both somatometric indicators; higher mean values
for height and weight in both genders as compared to national and regional mean values. Periodical surveillance of somatometric indicators represents amonitoring method for health and nutrition status during adolescence.
Keywords: postpuberty, physical development, health state

INTRODUCTION
Adolescence is an important period in human growth and maturation. Changes
now occurr with unique significance, now the adult earns many traits. Situated aroundbiological maturity and adult age, adolescence may offer the last opportunities for medical and social surveillance, for preventing major health problems of adults (1, 4).
Peripubertal stages (prepubertal period, 9-11 years old; early adolescence,
11-14 years old; postpubertal period, 15-19 years old) are runned trought under
the influence of biological and growth changes, but also of temperament andpersonality, social influences and mature expectations.
Postpuberty (15-19 years old) is defined by features described briefly
below.
Physical development- Most young people have entered or completed phase of puberty.
- Less variation in levels of physical growth and sexual maturation.
- Many young people have achieved adult height and other thresholds ofadult physical development.
Cognitive development- Vast majority of thinking skills: can think abstractly and hypothetically; can discern
the basic principles of phenomena and may apply them in new situations, can think about
the future, taking into account more possibilities and results of hypothetical events.
- A greater ability in perspective may result in empathy and consideration forothers, new interest in topics about society.
Moral development- Less self-centredness with aging. Increased empathy for abstract values
and moral principles.
- For some, increased skills in taking the perspective of others, can see society

more widely and appreciate the principles of laws, the "principal" morality.
- More proportions of cognitive and emotional development; the adolescentoften follows a value that he violates in the same time.
The concept of self
- The identity formation proces is intense. Experimentation of different roles:
appearance, sexuality, values, friends, ethnicity, occupations in particular.
- Some girls can live obesessive disorders in diet food, especially thoseoverweighted or those that have major family conflict relations and are chronic
depressed.
- Young people can explore various patterns of identity formation: strong ethnic
identity, bicultural identity ; assimilation into the majority culture or alienation fromthe majority culture.
Psychological and emotional traits- For some, increased ability to agree with others. Vulnerability to concernes,
depression, and care toward others, especially among girls.
- Many shows an increase in responsible behaviors.
Relations with parents and other adults- Conflicts with parents are more rare with age: enhanced ability to watch
their parents and taking their views into account, most maintain good relationships
with parents.
- Increased interest in assuming the responsibilities of „adult humans”: own
management accounts, washing their laundry, buying clothes, preparing meals,
the execution of repairs.
- Usually take most decisions themselves, preparing for their own family.
- They need to balance between time spent with adults and time spent with
their fellows.
- Continue to benefit from some parental limits and monitoring, often

Received April 2009. Accepted may 2009. Address for correspondence: Dr. Radu Bagiu, “Victor Babes” University of Medicine and Pharmacy Timisoara, No. 2A Eftimie Murgu Square, Timisoara 300041, Romania

24

CMYK

Fiziologia - Physiology  2009.19.2(62)(years)
15
16
17
18
19

objecting them.
- Usual conflicts because of money, imposed rules, domesticities, appearance
and activities with fellows.
Relations with fellows- Fellows help the youth to explore and develop their own identity.
- Friendships between the sexes become more common.
- The antisocial fellow groups may increase antisocial behavior.
- Close friendships help the youth in the development process of an own
identity, separate from that of a child in the family (2, 5).

_
Xp
171.0
173.0
175.0
177.0
177.0

Ĳ
7.0
6.8
7.1
6.5
6.8

_
Xp
60.3
62.6
65.5
68.0
68.4

Ĳ
6.8
7.0
7.2
6.9
7.0

Table II. Physical development level in girls of 15-19 years old

In the investigated age range, 15-19 years old, height and weight are chaAge
Height (cm)Weight (kg)racterized by
growth in both
sexes. The average values
for boys are always higher(years)__than the values correspondingtogirls.
Inboys,
theaverage
Xp
Ĳ
Xp heightĲ increased by 6
cm, and average
weight
by
8.1
kg.
In
girls,
averageheight increased
15
164.0
6.1
53.6
6.6 by 2 cm andaverage weight
by
2.3
kg.
16
164.0
6.7
54.9
6.7
17

165.0

6.2

54.6

6.2

2. The18
pace ofgrowth6.5
in height54.3
and weight
165.0
7.0 of boys
and girls19(Table166.0
III)
6.6
55.9
6.7

Table I. Physical development level in boys of 15-19 years old
Table III.
Growthofspeedsof height
weight means
in boysand girlsof 15-19
Table III. Growth
speedsheightandandweightmeans
in boys
andgirlsyearsof old15-19 years old
Age
Height (cm)Weight (kg)The present paper is an actuality maintenance assessment of current healthAge(years) Growth
speed of_
_ Growth speed ofin adolescence, through the study of the physical development as a direct indicator(years)heightXp means Ĳ(cm)Xpweight means
Ĳ (kg)of health state.The study that underlies the work continues the research of physicalGirls
15 Boys
171.0
7.0
60.3Boys 6.8 Girlsdevelopment of young people, performed in Romania at an interval of 7 years and
15/16 16
2.0
173.0
6.80
62.62.3
7.0 1.3
started in 1950 (7) and research in the Banat area (3, 4).
16/17 17
2.0
1.0
175.0
7.1
65.52.9
7.2 -0.3
17/18 18
2.0
177.0
6.50
68.02.5
6.9 -0.3
18/19 19
0
1.0
177.0
6.8
68.40.4
7.0 1.6
METHODOLOGY AND MATERIAL
Tabel IV. Distribution of percentage difference between the average values of height and weight in boys and g
Increased
speeds ofdevelopment
height inold,
boysonwere
2incmgirls
per yearbetweenyears
15 andold
18
years
ageofgroups
Research was conducted on a representative population of 2908 high school students
Table
II. Physicallevelof 15-19
yearsAgeold, and between
18 anddifference*
19 years old therePercentage
hasn’t been andifference*increase. In girls,
Percentage
from Timis county, urban area, 51.5% girls and boys 48.5% during 2003-2005.
Age(cm)
Weight(kg)
forboys-girls
weight,boys-girls
the(years)
increase was
onlyheight,
1 cm Height
between16
and 17 yearsfor
oldrespectively
18 and 19 years(years)_(%)_(%)
old; between 15-16 years old and 17-18 years old there hasn’t been an increase.
Xp
Xp +12.5Ĳ
15+4.2 in boysĲ ranged from
Increased speeds of weight
2.3 kgr and 2.9 kg per year
METHOD
15
164.0
6.1
53.6
6.6
+5.4 18-19 years old, the increase+14.2
aged1615-18 years
old,betweenwas
It was used the anthropometric method for determining the two main somato16
164.0
6.7
54.9 +19.9
6.7only 0.4 kg. In
17+6.0
girls, growth 17
velocities were
1.3
and
1.6
kg
between15-16
years
mertic indicators: height and weight (8). The individual measurements were statistically
165.0
6.2
54.6 +25.2
6.2old and 18-19
18+7.2
years old; at 1618years comporative
to 176.5
years and 1754.3
years comporative
165.0
7.0 to 18 years,
processed by computer, depending on the measured indicator, by age and sex criteria.
19
+6.6
+22.3
weight averages
were lower.
19
166.0
6.6
55.9
6.7(weight)girls - 1]
It was calculated the weighted arithmetic mean (Xp) and standard deviation (τ). There * Percentage
difference: 100x [height (weight) boys / height

Specific aspects of male and female gender in the investigated group, overlapwere appreciate: the level of increase of height and weight in both sexes; growth speed
Table V. Distribution of percentage
difference
betweenaveragehumanvaluesbody:of height
and weightof the inspectedping general
growthtrend inand
thethepostpuberty
increaseheightTable
speedsof height
weight means
in boysanandgirlsofof
15-19 years oldlevels of somatometric indicators in the range 15-19 years old. Average heightand III. Growthcomparedtotheguidancefor
Romania,
urban
area,
1999,
by
age
and
sex
criteria
and weight to the male gender, compared with female gender, growth speeds areweight values of the population taken in the study were compared with the guidanceAgeGrowthspeed of
speed
of of
Age
Percentage
differencesof meansrelatively
low, which orientates
towards
achieving
the Growth
futureheightand weightmeans for Romania, 1999, and the guidance means for Banat, 1998.
(years)heightmeans
weightmeans(years)height(%)(cm)weight(%) (kg)
future adults in postpuberty, earlier in girls compared with boys.

Girls
Boys
Girls
Boys
Girls
Boys
Girls
15/16
2.0
0
2.3
1.3
15
+1.0
+1.3
+10.0
+4.0
3. Correlation
RESULTS AND DISCUSSIONS
16/17
2.0 of average
1.0 values
2.9for height-0.3and
16
+0.1
+0.7
+4.1
+2.8
weight17/18
2.0 and sex
0(Table IV)
2.5-0.3
1. Physical development level (Tables I, II)17 with age
+0.1
+1.1
+3.6
+0.9
18/19
0
1.0
0.4
18
+0.4
+1.0
+3.8
-0.11.6
Tabel IV. Distribution of percentage difference between the average values of height andTabledevelopment
level
boys
of
15-19
yearsofold
Table
I. Physical developmentlevel inin boysof 15-19
years
old years
Tabel
IV.
Distribution
percentage
difference between
the average values/ ofheight and weight- in*oldPercentage
difference:
100x [height(weight)boys
height(weight)girls
1] boys and g
TableI.I.PhysicalPhysicaldevelopment
level
in
boys
of
15-19
weight in boys and girls of 15-19 years old, on age groups
years
old,
on
age
groupsAge
HeightWeight (kg)(kg)
Age
Height (cm)(cm)
WeightAge
Percentage difference* Percentage difference*(years)___(years)Table
difference
betweenthe average valuesof height
and weight of the inspecte_ VI. Distribution of percentage(years)for height,
boys-girls
for weight,
boys-girls
Xp
Ĳ
Xp
Ĳ
Xp
Ĳ
Xp
Ĳ
compared to the guidance for
Banat, urban area, 1998, (%)
by age and sex criteria
(%)
15
171.0
7.0
60.3
6.8
15
171.0
7.0
60.3
6.8
Age
Percentage
differences of means
15
+4.2
+12.5
16
173.0
6.8
62.6
7.0
(years)height(%)
weight
(%)
16
173.0
6.8
62.6
7.0
16
+5.4
+14.2
17
175.0
7.1
65.5
7.2
Boys+6.0 Girls
Boys +19.9 Girls
17
175.0
7.1
65.5
7.2
17
18
177.0
6.5
68.0
6.9
18
177.0
6.5
68.0
6.9
+0,4 +7.2 +0,1
+7,4 +25.2 +1,7
1815
19
177.0
6.8
68.4
7.0
19
177.0
6.8
68.4
7.0
-0,5 +6.6
0+0,4 +22.3 +1,8
1916
17
-0,6
+0,1
+0,7
* Percentage difference: 100x [height (weight) boys / height -2,5
(weight)girls - 1]
Table II. Physical developmentlevel in girlsof 15-19of
years
old
Table
girls
15-19
years old
old
18
+0,1100x [height+0,4-0,8* Percentage
difference:(weight) boys / height(weight)girls - 1]-4,5
TableII.
II.PhysicalPhysical development
development level
level inin girlsof 15-19
years
Table V. Distribution of *percentage
difference
the average values of/ height(weight)girls
height and weight of- 1]
Percentage
difference:
100x
Between
15-19between
years
old,[height(weight)boys
percentage
differences, calculated
to compare the the inspected
Age
HeightWeight (kg)(kg)
Age
Height (cm)(cm)
Weightcompared to the guidance for Romania, urban area, 1999, by age and sex criteria
height and weight corresponding to boys and girls, boys are positive and indicate(years)___(years)_Age
Percentage differences of means
Xp
Ĳ
Xp
Ĳ
an increased
to boys(%)
versus girls. This period
is after(%)
the second cross
Xp
Ĳ
Xp
Ĳ
(years)growing level
heightweight
15
164.0
6.1
53.6
6.6
correlation
curves
of
the
media
age
and
sex
between14
and15
years old. The
15
164.0
6.1
53.6
6.6
Boys
Girls
Boys
Girls
16
164.0
6.7
54.9
6.7
largestpercentage
difference
in
favor
of
boys,
were
calculatedforweight,
16
164.0
6.7
54.9
6.7
15
+1.0
+1.3
+10.0
+4.0 towards
17
165.0
6.2
54.6
6.2
height,
and
for
both
anthropometricindices,
in
the
age
range
17-19
years old. The
17
165.0
6.2
54.6
6.2
16
+0.1
+0.7
+4.1
+2.8
18
165.0
6.5
54.3
7.0
research17
confirms the+0.1
human ontogeny.
18
165.0
6.5
54.3
7.0
+1.1
+3.6
+0.9
19
166.0
6.6
55.9
6.7
19
166.0
6.6
55.9
6.7
18
+0.4
+1.0
+3.8
-0.1
* Percentage difference: 100x [height(weight)boys / height(weight)girls - 1]
and
means
in boysboys andand girls
girls ofof 15-19
15-19years
yearsold
oldeIII.
III.Growth
Growthspeeds
speedsof
ofheight
heightand weightweightmeans in25
2009.19.2(62) Fiziologia- Physiology
AgeGrowthspeedofGrowthspeedofAge
Growth speed of
Growthspeed ofof percentage difference between the average values of height and weight of the inspecte
Table
VI. Distribution(years)heightweight means
means (kg)(kg)
(years)height means
means (cm)(cm)
weightcompared to the guidance for Banat, urban area, 1998, by age and sex criteria
Boys
Girls
Boys
Girls
Boys
Girls
Boys
Girls
Age
Percentage differences of means
15/16
2.0
00
2.3
1.3
(years)
15/16
2.0
2.3
1.3
height (%)
weight (%) CMYK
16/17
2.0
1.0
2.9
-0.3
16/17
2.0
1.0
2.9
-0.3
Boys
Girls
Boys
Girls

16/17
2.0
1.0
2.9
-0.3
15/16
2.0
0
2.3
1.3
17/18
2.0
0
2.5
-0.3
16/17
2.0
1.0
2.9
-0.3
18/19
0
1.0
0.4
1.6
17/18
2.0
0
2.5
-0.3
ibution of percentage
difference0between the1.0
average values
18/19
0.4of height and1.6 weight in boys and girls of 15-19
years old,age groups
ibution of percentage difference between
theon
averagevalues of height and weight in boys and girls of 15-19
Age
Percentage
difference*
Percentage difference*years old, on age groups(years)for height, difference*boys-girls
for weight,difference*boys-girls
Age
Percentage
Percentage(%)(%)(years)for height, boys-girls
for weight, boys-girls
15
+4.2
+12.5
(%)
(%)
16
+5.4
+14.2
4.
Comparingtheaverage
growthlevel in theadolescents from Timis county in the last decade.
15
+4.2
+12.5
17
+6.0
+19.9
studiedpopulation,withthenational
andregional
16
+5.4
+14.2
18 (Table V, VI)
+7.2
+25.2
means
17
+6.0
+19.9
19
+6.6
+22.3
CONCLUSIONS
18
+7.2
+25.2
* Percentage
difference:
100x[height difference(weight)
boysthe/averageheight(weight)girls- 1] Rapid changes that occur in adolescence relates to height and weight augV. Distributionof percentage
betweenvaluesof height
19Table+6.6+22.3
and weight of the inspected consignment compared to the guidance for Romania, urban area,
Percentagedifference
difference:between
100x [height(weight)
boysof/ height (weight)girls
1]mentation,
definedby the term growth, and development towards adult status
ution of*percentage
the averagevaluesand weight of- theinspected
consignment
1999, by age and sex criteria
comparedtotheguidancefor
Romania,
urban
area,
1999,
by
age
and
sex
criteria
definedbytheterm
maturation.
ution of percentage difference between the average values of height and weight of the inspected consignment
Age
Percentage
differences
of
means
If
growthandmaturation
takes place in parallel to the same person, they cancompared to the guidance for Romania, urban area, 1999, by age and sex criteria(years)height(%)
weight(%)
bevery
different
fromone
individual
to another. Chronological unfolding of matuAge
Percentage differences of means
Boys
Girls
Boys
Girls
rationdiffers
fromahealthychild
toanother, from genetic considerations, under(years)height (%)
weight (%)
15
+1.0
+1.3
+10.0
+4.0
the influence of environmental factors and health care. It is difficult to distinguish
Boys
Girls
Boys
Girls
16
+0.1
+0.7
+4.1
+2.8
15
+1.0
+1.3
+10.0
+4.0
between normal variability by genetic and hormonal changes of adolescence, and
17
+0.1
+1.1
+3.6
+0.9
16
+0.1
+0.7
+4.1
+2.8
associated changes in environmental factors (6).
18
+0.4
+1.0
+3.8
-0.1
17
+0.1
+1.1
+3.6
+0.9
Periodic surveillance of the somatometric indicators in the basic and periodic* Percentage18difference:
100x [height(weight)boys
- 1]
+0.4
+1.0
+3.8/ height(weight)girls-0.1* Percentage difference: 100x [height(weight)boys / height(weight)girls - 1]

medical examinations from the children and adolescents collectivities, attaches* Percentage difference: 100x [height(weight)boys / height(weight)girls - 1]a particular importance to anthropometry in monitoring the state of health andution of percentageTable
difference
betweenthe average
ofaverageheightandweight of theinspected
VI. Distributionof percentage
difference values
between thevaluesof height
nutrition(8). consignment
andtoweightofthe inspectedfor
consignment
comparedto the guidancefor
Banat,
urban
area,
comparedtheguidance
Banat,
urban
area,
1998,
by
age
and
sex
criteria
ution of percentage difference between the average values of height and weight of the inspected consignment
Percentage
differencesof means
1998,
bythe
ageguidanceand sex criteriafor
compared
toAge
Banat, urban
area, 1998,
by age and sex criteria(years)height(%)
weight(%)
REFERENCES
Age
Percentage differences of means
Boys
Girls
Boys
Girls
1.
Miu N. Tratat de medicină a adolescentului, Casa Cărţii de Ştiinţă,(years)height (%)
weight (%)
Cluj Napoca, 1999.
15
+0,4
+0,1
+7,4
+1,7
Boys
Girls
Boys
Girls
2. Santelli JS, Rogers AS, Rosenfeld WD, et al. Guidelines for Adoles16
-0,5
0
+0,4
+1,8
15
+0,4
+0,1
+7,4
+1,7
cent Health Research. Journal of Adolescent Health 2003;33(5):39617
-0,6
+0,1
+0,7
-2,5
16
-0,5
0
+0,4
+1,8
409.
18
+0,1
+0,4
-0,8
-4,5
3. Vlaicu B. Dinamica dezvoltării fizice şi aspecte comportamentale
17
-0,6
+0,1
+0,7
-2,5
* Percentage18difference:
100x [height(weight)boys- 1]la şcolari. Editura Signata, Timişoara, 1994
+0,1
+0,4
-0,8 / height(weight)girls-4,5
4. Vlaicu B, Vasilov M. Adolescenţa. Particularităţi antropometrice* Percentage difference: 100x [height(weight)boys / height(weight)girls - 1]în Banat şi Moldova, Editura Eurobit, Timişoara, 1998.
* Percentage difference: 100x [height(weight)boys / height(weight)girls - 1]

For both investigated anthropometric indicators, averages calculated byage and sex were higher compared to the national indicatives, study stage 1999,
expressing the reality of an superior physical development in adolescents of 15-19
years old in western Romania. Compared with the height and weight means from
Banat, calculated in a national study conducted 10 years ago, prevail the higher
values from the current study; the lowest mean values recorded in one third of
the current results show a reduction in the rate of physical development at the

5. Williams PG, Holmbeck GN, Greenley RN. Adolescent health psychology. Journal of Consulting and Clinical Psychology 2002;70(3):828-842.
6. Woodruff BA, Duffield A. Anthropometric assessment of nutritional status in adolescent populations in humanitarian emergencies, European Journal of Clinical Nutrition 2002;56(11):1108-111.
7. ***, MS, ISPB. Dezvoltarea fizică a copiilor şi tinerilor, 0-18 ani,
1999.
8. ***, Utilisation et interpretation de l’anthropometrie, Serie deRapports techniques 854, 1995.

CORELAŢIA UNOR INDICI ANTROPOMETRICI CU VÂRSTA ŞI SEXUL ÎNPERIOADA DE POSTPUBERTATE
REZUMAT
Postpubertatea, cu limite între 15-19 ani, este dominată de reducerea vitezelor de creştere ale taliei şi greutăţii, odată cu instalarea pubertăţii.
S-a luat în studiu o populaţie reprezentativă 2908 adolescenţi timişeni de 15-19 ani. Metoda de lucru a fost antropometria. Rezultatele studiului
au evidenţiat o creştere taliei şi greutăţii la ambele sexe; valori medii superioare la băieţi, comparativ cu fetele la ambii indicatori somatometrici;
valori medii mai mari pentru talie şi greutate, la ambele sexe, faţă de mediile orientative naţionale şi parţial, pentru cele zonale. Supravegherea
periodică a indicatorilor somatometrici se constituie în metodă de monitorizare a stării de sănătate şi de nutriţie în adolescenţă.
Cuvinte cheie: postpubertate, dezvoltare fizică, stare de sănătate

26

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Fiziologia - Physiology  2009.19.2(62)LYCOPENE INVOLVEMENT IN HEPATIC LIPID DISORDERS
DUE TO HYPERTHYROIDISM IN RATSADELA ELENA JOANTA1, SANDA ANDREI2, NICOLETA DECEA1, ADRIANA MURESAN1, REMUS
MOLDOVAN1, OLIVERA STANOJLOVIĆ3, DRAGAN DJURIC3, MANOLE COJOCARU4
1
Department of Physiology, University of Medicine and Pharmacy, Cluj-Napoca, Romania
2
Department of Biochemistry, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania
3
Laboratory of Neurophysiology, Institute of Medical Physiology “Richard Burjan”, School of Medicine, University of Belgrade, 11000 Belgrade, Serbia
4
Colentina Clinical Hospital, Clinical Immunology Department, Bucharest, Romania

ABSTRACT
Recent studies showed the relationship between oxidative stress and thyroid disorders. Effect of lycopene, the main carotenoid in tomato, was assessed onoxidative stress and on lipid disorders generated by excess in L-Thyroxin in hepatic tissue. Experiments were performed on white, male, Wistar rats. Lipids
were extracted with methanol and chloroform, according to the Folch procedure. Fatty acids analysis was performed using gas chromatography coupledwith mass spectrometry. Lipid peroxides level was assessed by measuring the production of thiobarbituric reactive substances, according to the method of
Buege and Aust. A diet rich in lycopene led to an increase in the fatty acids content, especially in the ratio between unsaturated fatty acids and saturated fatty
acids, showing its antioxidant effect. Lipid peroxides increased in rats treated with L-Thyroxin, due to the oxidative stress exerts by the thyroid hormone, anddecreased in rats fed a diet rich in lycopene. Lycopene supplementation revealed a reduction in lipids susceptibility to oxidative injury due to the L-Thyroxin
administration. Carotenoids association to classic treatment of the hyperthyroidism could be recommended.
Key words: lycopene, oxidative stress, thyroid hormones, lipid disorder, liver

INTRODUCTION
In recent years, it was noted an increasing prevalence and incidence of thyroid
disease. Our understanding of the effects of thyroid hormones under physiologicalcircumstances, as well as in pathological conditions, has increased dramaticallyduring the last two centuries and it has become clear that overt thyroid dysfunction
is associated with significant morbidity and mortality. Both hypo-and hyperthyroidism and their treatments have been linked with increased risk from cardiovasculardisease and the adverse effects of thyrotoxicosis in terms of osteoporosis risk arewell established (6, 25).
Thyroid hormones play a crucial role in the regulation of mitochondrial oxidative metabolism; the synthesis and degradation of proteins and vitamins, such asvitamin E, vitamin A, and ß-carotene; the sensitivity of tissues to catecholamines;
and the regulation of antioxidant enzyme levels (2, 9). Overt hyperthyroidism andhypothyroidism represent opposite clinical conditions characterized respectively byenhanced oxidative metabolism and reduced lipid and lipoprotein plasma levels
and by reduced oxidative metabolism and markedly increased lipid and lipoprotein
plasma levels. The hypermetabolic state that characterizes hyperthyroidism should
accelerate free radical production in the mitochondria and induce changes in theantioxidant defense system (2).
Accumulating evidence has suggested that the hyper metabolic state in
hyperthyroidism is associated with increased reactive oxygen species production
and lipid peroxidation products in some of the rat’s tissues. The response of theantioxidant systems to hyperthyroidism is unclear. Changes in the nonenzymatic
antioxidants levels (α-tocopherols, retinol, β-carotene, coenzyme Q) and the antioxidant enzymes activities in various tissues were found to be imbalanced andoften opposite (24).
Lycopene, the main carotenoid in tomato, has been shown to be a potent
antioxidant in vitro and in vivo. In vitro studies showed that lycopene has the highest

ability to quench singlet oxygen and trap peroxyl radicals (20). In human and animal
body, lycopene is able to protect cellular biomolecules against oxidative damages;
therefore, it is proved to have beneficial effects in wide range of ROS mediateddiseases, including cancer (1). Steel, there is a lack of information regarding theno enzymatic antioxidants effect in hyperthyroidism.
The purpose of our study was to investigate the lycopene involvement in lipid
disorders generated by an excess in L-Thyroxin, in rat hepatic tissue.

MATERIALS AND METHODS
Animals and housing conditions
Wistar rats, male, 90 days old, weighting 180-330g, were maintained underpathogen-free conditions in a temperature-controlled (23 ± 10C, 50–70% relative
humidity) and light-controlled (illuminated from 0600–1800 h) room. None of theanimals died unexpectedly.
Experimental groups
Three groups of animals, each group consisting in 10 rats, were investigated,
as follows:- Group I: untreated animals (control) fed a standard diet
- Group II: L-Thyroxin injected rats L-thyroxin (10µg/100 g body weight/day),intraperitoneal administered fed a standard diet
- Group III: L-Thyroxin injected rats fed a standard diet rich in lycopene(tomatoes powder).
Distilled water was available to all animals ad libitum.
Experiments were performed for 7 days.
All animal studies were done according to the local guidelines for animal
research andprincipals of the European Convention for the Protection of Vertebrate Animals

Address for correspondence: Dr. Adela Elena Joanta MD, PhD, Department of Physiology, “Iuliu Hatieganu” University of Medicine and Pharmacy, Emil Isac Street No. 13, 400023 Cluj- Napoca, Romania, Phone: 0040
745 512428, Fax: 0040 264 598575, e-mail: adelaelena@yahoo.com

2009.19.2(62)  Fiziologia - Physiology

27

CMYK

RESULTS
Lipid peroxidation
Malondialdehyde levels in hepatic tissue of the thyroxin treated rats were foundsignificantly increased (P<0.001) as compared to control values. Administrationof both thyroxin and lycopene (group III) led to a significant reduction (P<0.001)
of the lipid peroxides as compared to control values (Table I).
Table I. Lipid peroxides level (µmols malondialdehyde/mg protein) in rat liver

Experimental groups
C (n=10)
T (n=10)TL (n=10)

MDA
4.01 r 2.25
10.85 r 1.85 *5.99 r 3.84 **determined a significant increase (P<0.001) in the total fatty acids level in rat
liver as compared to control group. Also, lycopene supplementation led to a
Experimentalgroups of the same parameter asMDA
significant
increase (P<0.001)
compared to thyroxinC(n=10)
4.01 r 2.25
treated animals (Table II).
T (n=10)10.85 r 1.85 *TL(n=10)
5.99
Table II. Fatty acids content (mg/g tissue) in the rat liver r 3.84 **Experimental groups
ExperimentalgroupsC (n=10)C(n=10)
T (n=10)
T (n=10)TL(n=10)TL (n=10)

Dynamicof unsaturated fatty
acids/C (n=10)1.75 saturated
3.95r
**
fatty
acids
ratio
in
hepatic
tissueT(n=10)
34.03
r
3.66
*
3
Lipid peroxidation
the ratio between unTL (n=10)is assessed, also, by measuring
12.81r 2.66*** This ratio increased in lycopene
saturated fatty acids and saturated fatty acids.
2.5
group due to its antioxidant effect, as compared to control and thyroxin treatedrats (Figure1).

UFA/SFA

3.52

CMYK
2.5

*

4
3.5
3
2.5
4
2
3.5
1.5

*

2.51

3
1
2.5

0.5
2

0.5
2

1.50

0
C

T

TL

1

1.5
1

Experimental groups
0.5 Fatty acids (%)
C (n=10)
T (n=10)TL (n=10) 0.5
Palmitoleic acid4r2.25 *7.5r2.25 **1.5r0.75
Linoleic
acid
0
7.5r3.5
20r3.50 *25r3.75 **0
ArachidonicCacid
10r2.75T 18.5r2.25 * TL23r1.5 **Eicosatrienoic acid8r1.75 *4r1.75 **2r0.25
Fig. 1. Dynamic of unsaturated fatty acids/ saturated fatty acids ratio in rat hepatic
Oleic
acid
22r3.75*
7.5r4.5**
13r3.25
tissueExperimental groups
Legend: UFA unsaturated fatty acids; SFA saturated fatty acids; C control group; T thyroxin
Fatty
acidsTL thyroxin(%) and lycopene
C (n=10)(n=10)TL (n=10)
treated group;
co-treated group;T* p<0.001 thyroxin vs control;** p< 0.001
Palmitoleic
acid
4r2.25*
7.5r2.25**
1.5r0.75
thyroxin vs thyroxin+lycopene
Linoleic acid
7.5r3.5
20r3.50 *25r3.75 **Arachidonic
18.5r2.25
23r1.510r2.75 from
Fattyacidacids percentage
lipid* extractin**rat
Eicosatrienoic
acid
8r1.75*4r1.75**2r0.25
hepatic tissue
Oleic acid22r3.75 *acids content
7.5r4.5
13r3.25 and eicosatrienoic
Palmitoleic, linoleic, arachidonic
was**sig-

nificantly higher (P<0.001) in both groups: thyroxin, respectively thyroxin andlycopene treated rats as compared to control. Regarding the oleic acid content, it
was noticed a significant increase determined by thyroxin and noticeably decrease(P<0.001) due to lycopene administration as compared to control and thyroxin
treated groups. (Table III)Regarding the lipid extract from the hepatic tissue, thyroxin administration

3

**

1.5
3

tissue)C (n=10)
3.95r 1.75
Fatty acids
content of the lipid
extract
T (n=10)34.03
r 3.66in* the rat
liver
TL (n=10)
12.81r 2.66****

tissue)

3.5

Legend:MDA malondialdehyde;
C control group;
T thyroxin
treatedgroup;(mg/g
TL thyroxin and lycoExperimentalgroups
Total
fatty
acids
pene co-treated group; * p< 0.001 thyroxin vs control; ** p< 0.001 thyroxin vs thyroxin+lycopene

3.5 28

Total fatty acids (mg/gtissue)
MDA1.75
3.95r
4.01
34.03rr2.25
3.66 *
r 1.85*
10.85
12.81r
2.66**
5.99 r 3.84 **Legend: C control group; T thyroxin treated group; TL thyroxin and lycopene co-treated group;* p< Experimental
0.001 thyroxin vs control;** p< 0.001 thyroxin
vs thyroxin+lycopene
groupsTotalfatty acids (mg/g

UFA/SFA

Used for Experimental and Other (published in the Official Daily N.L. 358/1-358/6,
18, December 1986) and according to The UFAW Handbook on the Care andManagement of Laboratory Animals published by Blackwell Science (www.tiny.
cc/9y7Sa).
Experimental procedures
Animals were sacrificed by decapitation under ether anesthesia in the 8 th
day of the experiment. The liver was quickly excised and placed into Petri dishes
containing ice-cold isolation medium. Lipid extracts were obtained with methanoland chloroform, according to the Folch procedure (12). Fatty acids analysis was
performed using gas chromatography coupled with mass spectrometry (7). Level oflipid peroxides was evaluated fluorometrically as thiobarbituric reactive substances(TBARS), according to the Satoh method (22).
Quantitative lycopene determination
Tomatoes powder is a natural product made by Künding‑Food Company as
nutritive supplement. Quantitative lycopene determination was assessed by extraction procedure, followed by spectrophotometer measurement. Average lycopene
content in tomatoes powder was 125g-lycopene/2g tomatoes powder. Rats received
0, 4 g tomatoes powder / 100 g body weight / day.
Lipid peroxidation assay
Level of lipid peroxides was evaluated fluorometrically as thiobarbituric reactive substances (TBARS), according to the Satoh method (22). Fluorescent reaction
products were estimated spectrofluorometrically at 515 nm excitation and 553 nm
emissions using a Kontron SFM25 spectrofluorometer. Results were expressed asµmoles malondialdehyde (MDA) per milligram of protein (23).
Protein extract assay
Liver samples were washed with physiological serum buffered with potassium
phosphate (10 mM), pH: 7, 4. Proteins were extracted using a potassium phosphate
solution (50 mM), pH: 7, 35 (7).
Total protein amount was photometrically assessed, using Robinson-Hogden
method for plasma, applied for animal tissues (7).
Statistical analysis
Data are reported as mean ± SEM (standard error of the mean). Data wereanalyzed by Student “t” test. Statistical significance was defined as P<.05.

Table III. Fatty acids percentage from lipid extract in rat hepatic tissue
4
3.5
3

Fiziologia - Physiology  2009.19.2(62)

1.5

1.5

1

1

0.5

0.5

0

0
C

T

TL

Legend: C control group; T thyroxin treated group; TL thyroxin and lycopene co-treated group;

Fatty acids (%)
Palmitoleic acid
Linoleic acidArachidonic acid
Eicosatrienoic acid
Oleic acid

C (n=10)
1.5r0.75
7.5r3.5
10r2.75
2r0.2513r3.25

Experimental groups
T (n=10)TL (n=10)
4r2.25 *7.5r2.25 **20r3.50 *25r3.75 **18.5r2.25 *23r1.5 **8r1.75 *4r1.75 **22r3.75 *7.5r4.5 *** p< 0.001 thyroxin vs control; ** p< 0.001 thyroxin+lycopene vs control

DISCUSSION
Acceleration of the basal metabolic rate and the energy metabolism of tissues
in several mammalian species represent one of the major functions of the thyroid
hormones (26). The liver is the place where tetra-iodothyronine provides the metabolic
thyroid hormone: tri-iodothyronine through deiodination by deiodase type I (18).
Much of the reactive oxygen species production occurs in mitochondria, via
oxidative phosphorilation. Because the mitochondria contains specific receptors forthe thyroid hormones, being one of the “favorite” target for them, the concept about
a possible relationship between reactive oxygen species production and thyroid pathology has increasing importance (15,16). When the thyroid hormones production
increases, hepatic tissue is, also, subjected to oxidative stress because of their action
on liver mitochondria and on Kupffer cells (8).
'Moreira and coworkers (17) evaluated the influence of tomato powder indiets differing in energy level on antioxidant status in blood and liver of rats. Foodintake and thiobarbituric acid-reactive substances contents in liver and plasma weresignificantly decreased by tomato powder at both energy levels. After tomato powder
supplementation, the hepatic levels of ubiquinol 9, alpha-tocopherol, lycopene andbeta-carotene were significantly enhanced. In plasma, only the contents of lycopene
and beta-carotene were enhanced. The erythrocytic and hepatic activities of catalase
were lower, while those of glutathione peroxidase were higher after the ingestionof tomato powder. Total and reduced glutathione contents in liver showed lower
levels in cafeteria-fed rats compared to the hypo energetic diet. This data suggestthat the lycopene and beta-carotene component in the tomato power supplement
might be beneficial for the prevention of oxidative damage in rats fed both types ofenergetic diets.
The effects of tomato product supplementation, containing lycopene, on
biomarkers of oxidative stress and carcinogenesis were investigated in human clinical
trials. Supplementation of tomato products, containing lycopene, has been shown tolower biomarkers of oxidative stress and carcinogenesis in healthy and type II diabetic
patients, and prostate cancer patients, respectively. Processed tomato products liketomato juice, tomato paste, tomato puree, tomato ketchup and tomato oleoresin havebeen shown to provide bioavailable sources of lycopene, with consequent increases inplasma lycopene levels versus baseline. Dietary fats enhance this process and should
be consumed together with food sources of lycopene. The mechanisms of action
involve protection of plasma lipoproteins, lymphocyte DNA and serum proteins against
oxidative damage, and anticarcinogenic effects, including reduction of prostate-specificantigen, upregulation of connexin expression and overall decrease in prostate tumor
aggressiveness. There is limited in vivo data on the health benefits of lycopene alone.
Most of the clinical trials with tomato products suggest a synergistic action of lycopene
with other nutrients, in lowering biomarkers of oxidative stress and carcinogenesis.
So, consumption of processed tomato products, containing lycopene, is of significant
health benefit and can be attributed to a combination of naturally occurring nutrientsin tomatoes. Lycopene, the main tomato carotenoid, contributes to this effect, but its
2009.19.2(62)  Fiziologia - Physiology

role per se remains to be investigated (4).
Our results could be related to the mentioned data: thyroxin administration
increased notably the malondialdehyde concentration in hepatic tissue. Lycopene, awell-known antioxidant, exerted a protected effect against oxidative stress induced
by an excess in thyroxin.
Thyroid hormones interfere with lipid biosynthesis, recruitment and degradation,
the last one being more potent than the first one. The specific effects of the thyroid
hormones on lipid metabolism consist in intensifying the triglycerides biosynthesis
and recruitment from the adipose tissue, increasing the free fatty acids level anddecreasing the lipoprotein-lipases activity (19). According to the Heimberg and his
coworkers, the fat acids biosynthesis, cetogenesis and, paradoxically, the fat acids
oxidation are intensified in hyperthyroidism. Also, the fatty acids esterification to
triglycerides is diminished (14).
The most significant peroxidation substrate is represented by the polyunsaturated
fatty acids from membranes. Lipid peroxidation is assessed, also, by measuring theratio between unsaturated fatty acids and saturated fatty acids (10). In our experiment,
this ratio decreased in thyroxin treated rats, due to lipid peroxidation intensifying, but
it increased in lycopene group due to its antioxidant effect.
Regarding the lipid extract from the hepatic tissue, thyroxin administration
determined a significant increase in the total fatty acids level as compared to control
group. Also, lycopene supplimenation lead to a significant increase of the same parameter as compared to thyroxin treated animals. These alterations are in concordancewith the results of Blenneman and coworkers: thyroid hormones enhance fatty acids
biosynthesis in the hepatic tissue (5).
Also, Thyroxin administration increased fatty acids biosynthesis, and lycopene
protected them against oxidation. By cumulating of these two effectes, it could be
explained the noticeable increased in fatty acids content: 8 times over the control.
Also, the lycopene protective effect is shown by the way in which the ratio between
unsaturated fatty acids and saturated fat acids was changed.
Fatty acids content varies accordingly to the thyroid hormones doses and to
the administration period. For instance, Faas and Carter showed that high doses oftriiodothyronin (over 25 µg/100g body weight), administered in rats for three weeks,determined a significant increase in stearic and arachidonic acid content and a decreasein palmitic, palmitoleic, linoleic and eicosatrienoic acid content of the hepatic tissue.
At lower triiodothyronin doses, the content in palmitic, palmitoleic and linoleic acidslightly increased. These disorders regarding the fatty acids content are caused by adecreased in D-6-desaturase activity, an enzyme involved in nonsaturated fatty acids
synthesis, with more than 25% (11).
In our study, low doses of L-Thyroxin administration (10 µg/100g body weight) fora week lead to a markedly increased in palmitic, oleic, linoleic and eicosatrienoic acids
content and to a noticeably decrease in stearic acid in rat liver. Venditti and coworkers (24)studied the lipid content of the hepatic mitochondria and microsoms in hyperthyroid rats.
The phospholipids content increased with 14% in mitochondria and decreased with 23%
in microsoms. It did not notice changes regarding the microsomal phospholipids content infatty acids but it decreased the content in linoleic acid and increased the level in arachidonic
acid in mitochondria.The most noticeable alterations were shown regarding the cardiolipids:
a 32, 4% increase in palmitic and stearic acid content concomitantly with a decrease in linoleic
acid level. Other researchers (31) investigated the alteration of the lipid pattern in rat-liver
mitochondria and microsomes induced by triiodothyronine administration. In mitochondria,
a 25% total cholesterol decrease and a 14% phospholipids increase have been detected. In
these hyperthyroid rat liver organelles, a strong decrease in the total cholesterol/phospholipids
molar ratio occurs. On the contrary, in microsomes from the same animals, a decrease of about
23% has been measured for both total cholesterol and phospholipids; hence, in this fraction,
the total cholesterol/phospholipids molar ratio is unaffected by hyperthyroidism. The liver
mitochondrial phospholipids composition, unlike the microsomal composition, is altered
29

CMYK

significantly in hyperthyroid rats; a 7.4% phosphatidylcholine decrease is accompanied bya similar additive percentage increase of both phosphatidylethanolamine and cardiolipin. In
regard to total phospholipids fatty acid composition in liver microsomes from hyperthyroid
rats, no variation has been observed compared with the control rats, whereas in mitochondria
from the same animals, a meaningful linoleic acid decrease with a similar arachidonic acidincrease has been found. In addition to fatty acid alteration, the separated mitochondrial phospholipids classes also exhibit some increase in stearic acid. Among phospholipids, cardiolipinchanges the most of the esterified fatty acids in hyperthyroid rat liver. In this compound, astrong increase in the percentage of both palmitic and stearic acid and a 32.4% decrease oflinoleic acid has been found.

CONCLUSIONSThis study demonstrated the oxidative stress and lipid disorders occurrence
in rat hepatic tissue, caused by thyroxin administration. The lycopene rich dietincreased the fatty acids content as compared to thyroxin administration. Also, thelycopene enhanced the ratio between unsaturated fatty acids and saturated fatty
acids, showing its antioxidant effect.
Lycopene supplementation revealed a reduction in lipids susceptibility tooxidative injury due to the L-Thyroxin administration. Carotenoids association toclassic treatment of the hyperthyroidism could be recommended.
Acknowledgements
We thank Prof Dr Victoria Ciurdaru for her help at lycopene determination.
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2005;11:123-129.
16. Joanta AE, Filip A, Clichici S, Andrei S, Daicoviciu D. Iodide excess
exerts oxidative stress in some target tissues of the thyroid hormones. Acta Physiologica Hungarica 2006:93:347-359.
17. Moreira EA, Fagundes RL, Filho DW, Neves D, Sell F, Bellisle F, Kupek E. Effects of diet energy level and tomato powder consumption
on antioxidant status in rats. Clinical nutrition 2005;24:1038-46.
18. Nagaoki T, Kaptein E, Berry MJ. Structure-Activity Relationships
for Thyroid Hormones Deiodination by Mammalian Type I Iodothyronine Deiodinases. Endocrinology 2000;138:213-219.
19. Pucci E, Chiovato L, Pinchera A. Thyroid and lipid metabolism.
International journal of obesity and related metabolic disorders
2000;22:109-112.
20. Riso P, Pinder A, Santangelo A, Porrini M. Does tomato
consumption effectively increase the resistance of lymphocyte
DNA to oxidative damage. The American journal of clinical nutrition
1999;69:712-718.
21. Ruggiero FM, Landriscina C, Gnoni GV, Quagliariello E. Lipid
composition of liver mitochondria and microsomes in hyperthyroid
rats. Lipids 1984;19:171-8.
22. Satoh K. Serum lipid peroxide in cerebrovascular disorders
determined by a new colorimetric method. Clinica chimica acta
1978;90:37- 43.
23. Simon BC, Cunningham LD, Cohen RA. Oxidized low density
lipoproteins cause contraction and inhibit endothelium-dependent
relaxation in the pig coronary artery. The journal of clinical investigation 1990;87: 75-79.
24. Venditti P, Balestrieri M, Salineo T, De Leo. Effect of thyroid state on
lipid peroxidation, antioxidant defences and susceptibility to oxidative stress in rat tissues. Journal of Endocrinology 1997;67:151-157.
25. Zhang J. The Mechanism of Action of Thyroid Hormones. Annual
Review of Physiology 2000;62:439-466.
26. Williams Textbook of Endocrinology: ed. 9, pp 435- 456.Eds WB
Sounders Company, Philadelphia, 1998

EFECTUL LICOPINEI ASUPRA TULBURARILOR LIPIDELOR LA NIVELHEPATIC INDUSE DE HIPERTIROIDISM LA SOBOLANIREZUMAT

Cercetari recente au evidentiat legatura dintre stresul oxidativ si disfunctia glandei tiroide. In acest studiu s-a urmarit efectul licopinei, principalul carotenoid
din tomate, asupra stresului oxidativ si tulburarilor lipidelor generate de excesul de tiroxina la nivel hepatic. S-au luat in studiu sobolani albi, sex masculin,rasa Wistar. Dieta bogata in licopina a determinat cresterea continutului in acizi grasi , in special a ratiei dintre acizii grasi nesaturati si cei saturati, subliniind
efectul antioxidant al acesteia. Concentratia peroxizilor lipidici a crescut in urma administrarii tiroxinei datorita stresului oxidativ indus de hormonul tiroidiansi a scazut la animalele ale caror dieta a fost imbogatita cu licopina. Suplimentarea dietei cu licopina a diminuat susceptibilitatea lipidelor la atacul oxidativ
indus de administrarea tiroxinei. In concluzie, recomandam asocierea unei diete bogate in carotenoizi la tratamentul clasic al hipertiroidismului.
Cuvinte cheie: licopina, stres oxidativ, hormoni tiroidieni, lipide, ficat

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Fiziologia - Physiology  2009.19.2(62)LARYNGEAL PAPILLOMATOSIS MANAGEMENTDELIA HORHAT, RALUCA MOCANU, MARIOARA POENARU, S. COTULBEA, OLIVIA TOMA,
L. FARA, C. SARAU, F. HORHAT, N. BALICA, MIHAELA PRODEAENT Department, ”Victor Babes” University of Medicine and Pharmacy Timisoara

Abstract
Objective: Juvenile recurrent laryngeal papillomatosis (RLP) is a viral disease caused by Human Papilloma Virus type 6 and 11, with an incidence of 4-7 cases/1
million people characterized by multiple recurrences of benign tumours of the larynx mucosa and significant morbidity on paediatric patient and strain on
their families. It represents the most frequent benign tumour of the larynx with a high morbidity, difficult to treat due to its recurrences and respiratory tract
dissemination. The aim of this study was to evaluate the results of combined treatment with CO2 laser microsurgery and interferon (IFN) alpha 2b in children
suffering from laryngeal papillomatosis.
Materials and Method: In the study were included, during a 13 years period (1996-2008), 28 patients aged from 2,9 to 9 years diagnosed with laryngeal papillomatosis. CO2 laser microsurgery (microsurgical excision and progressive vaporization of papillomas) followed by IFN adjuvant therapy was the management
applied. Tracheotomy was not necessary in any of the cases.
Results: Clinical examination revealed regression of papillomas in all patients. 19 patients (67.85%) had complete regression after primary CO2 laser microsurgery and additional IFN treatment, and 9 patients (32.15%) had partial response to the combined treatment. In these cases second intervention wasneeded. We encountered no perioperative or postoperative complications or severe side-effects.
Conclusions: The method of choice in laryngeal papillomatosis in children is the combination of CO2 Laser microsurgery and additional IFN treatment, improving the prognosis of this recurrent viral disease. Reachable functional results, good tumour control, short hospitalization makes it a favourable treatment inpaediatric patients.
Key words: juvenile laryngeal papillomatosis, CO2 laser treatment, IFN alpha 2B

INTRODUCTION
In 1871, MacKenzie noted the frequent association of skin warts and laryngeal
papillomas. Ullmann in 1923 was the first to verify an infectious etiology by injecting
homogenized papillomata from a child’s larynx into his own forearm and observing
the development of papillomata there (1).
It was not, however until 1956 that a paediatrician made an association
between maternal condylomata and the risk of childhood infection (2). In
1973 an intranuclear icosahedral virus was identified in lesions by electron
microscopy (3), and in 1980 human papillomavirus (HPV) DNA was identifiedin papillomata (4). Surgical debulking was advanced with the use of the CO2
laser and suspension microlaryngoscopy in 1972 (5) which remains the stateof the art. Current areas of promise include the use of drugs to slow the diseaseprogression. Originally called juvenile laryngeal papillomatosis, the disease has
been increasingly recognized in adults and generally goes by the name recurrent
respiratory papillomatosis (RRP). The human papilloma virus (HPV), has morethan 60 subtypes identified; 20 of these subtypes affect the epithelium of thegenital tract. Condylomata acuminata (subtypes 6 and 11) and cervical dysplasia(subtypes 16, 18, 31, 33 and 35) are the most common clinical manifestationsof HPV infections (6,7,8).
The human papillomavirus is a naked, double-stranded, icosahedrally-shaped
virus with circular supercoiled DNA that belongs to the Papovavirus family. "Papova"
is an acronym for the three types of viruses in the family – papillomavirus, polyoma-

virus, and simian vacuolating virus. There are only three papovaviruses pathogenic
to humans: HPV, JC and BK viruses which are polyoma viruses (JC virus has been
implicated in progressive multifocal leukencephalopathy and BK virus has been
isolated from urine of kidney transplant recipients).
Juvenile recurrent laryngeal papillomatosis (RLP) is extremely aggressive andresistant to treatment, usually surgical (9,10). It typically involves the trachea, but
may spread to the oesophagus and bronchi, and rarely, to the lung where it actuallydestroys tissue, dramatically worsening the prognosis. Although rare, it is the mostcommon benign tumour of the larynx (11, 12, 13).
The incidence of RLP has been reported as 1:1500 live births. RLP has a bimodal
age distribution and presents most commonly in children younger than 8 years(juvenile-onset recurrent respiratory papillomatosis [JORRP]) or in persons in the
fourth decade of life (adult-onset recurrent respiratory papillomatosis [AORRP]).
JORRP is more common and more severe than AORRP.

MATERIALS AND METHOD
Between 1.01.1996 – 31.12.2008, 28 patients with Juvenile recurrent laryngeal papillomatosis (RLP) have been treated in the ENT Department Timişoara. The
mean age at diagnosis was 4,9 years. The proportion of Juvenile recurrent laryngeal
papillomatosis cases diagnosed between 2,9 and 3,9 years old was 13,04% (4cases), from 4 to 4,9 years old 65,21% (17 cases), from 5 to 5,9 years old 8,69%

Received April 2009. Accepted may 2009. Address for correspondence: Dr. Delia Horhat, ENT Department, “Victor Babes” University of Medicine and Pharmacy Timisoara,
No. 2A Eftimie Murgu Square, Timisoara 300041, Romania
2009.19.2(62)  Fiziologia - Physiology

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(3 cases), respectively from 6 to 8 years old 13,04% (4 cases) (Figure 1).

11%

14%

14%

Hoarseness23

4

Voice changes

12

Weak cry9

Choking episodes

18

Foreign body sensation

17

Chronic cough61%

2,9-3,9 years
4-4,9 years
5-5,9 years
6-9 years

11

Dyspnoea

20

Inspiratory wheeze

13

Stridor
0

5

10

15

20

25

Fig. 1. Age distribution
Fig. 3. Presentation symptoms

The Juvenile recurrent laryngeal papillomatosis (RLP) affects males andfemales in approximativelly equal number: 15 cases (52,17%) were females, and13 males (48,83%) (Figure 2).

14%

11%

46%

54%
Females

Males

Fig. 2. Gender distribution

75%
CV, anterior commisureCV, subglotic region
CV, VF, epiglottis, ant. and post. commisureFig. 4. Papillomas Localisation

The most common presentation of RLP was hoarseness 22 cases (95,65%),
voice changes occurred in 1 case (4,34%), young children also presented weak cry
11 cases (47,82%), choking episodes 9 cases (39,13%), foreign body sensationin the throat 18 cases (78,26%), chronic cough 15 cases (65,21%), dyspnoea
11 cases (47,82%), inspiratory wheeze 20 cases (86,95%) and stridor 13 cases(56,52%) (Figure 3).
The diagnosis was made upon the visualization of warty excrescences and confirmed by biopsy. Histological there was an epithelial projection with a fibrovascularcore, and there is associated parakeratosis, koilocytosis, and acanthosis.
Regarding localisation of the papillomas 21 cases (75%) presented papillomas
at the level of vocal cords, and anterior commisure, 4 cases (14%) at the level of
vocal cords and subglottic region and 3 cases (11%) at the level of vocal cords,
anterior and posterior commisure, ventricular folds and laryngeal surface of theepiglottis. (Figures 4, 5, 6, 7)

Fig. 5. Intraoperative view

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Fiziologia - Physiology  2009.19.2(62)of therapy are to relieve airway obstruction, improve voice quality, and facilitate
remission. When the papillomas formations were disseminated on a large surface
and small in dimension (21 cases) localised at the level of vocal cords and anterior
commisure each formation was vaporised with CO2 laser, taking care to not involve
the vocal cord mucosa. In case of large and obstructive papillomas formations (4cases; at the level of vocal cords, anterior and posterior comisure, ventricular folds
and laryngeal surface of the epiglottis), we used a forceps, without touching the
mucosa or free edge of vocal cord. Regarding posterior commisure and subglottic
area (3 cases), the papillomas vaporisation was performed without intubation tube.
This area is vaporised last, under a good haemostasis. (Figures 8, 9, 10).

Fig. 6. Intraoperative viewFig. 8. Intraoperative viewFig. 7. Intraoperative view

Case management consisted of CO2 laser microsurgery followed by adjuvant
therapy with IFN alpha 2b. All patients underwent microsurgical excision and progressive vaporization of papillomas with the CO2 laser under general anaesthesia with good
exposure of the anterior and posterior commisure. It was very important to preserve theanterior commisure in order to prevent anterior stenoses, which are not rare.Tracheotomy
was not necessary in any of the cases. The dosage of IFN alpha 2b was 100,000 IU/kg/
day, 5/7 days during a period of 11 month followed by 50,000 IU/kg/day, 5/7 days for 1
month. Functional aim was to preserve a functional glottis and to protect the laryngeal
fundamental functions, for increased quality of patient’s life. Follow-up period varied
between 4 and 48 month (average 14 months). Followed up data were available for all
patients. All were followed up using a precise postoperative protocol.

RESULTSOne goal is to eradicate disease without damaging normal structures. Traditionally this has been done with either cold steel or with the CO2 laser. Other goals
2009.19.2(62)  Fiziologia - Physiology

Fig. 9. Postoperative view

Treatment involves repeated debulking of the warty growths by laser surgery.
Care must be taken especially around the anterior and posterior commisures toavoid the formation of webs. It is better to be more conservative on one side andleave behind disease than to be aggressive and develop a web. Care must be taken
to protect the operating room personnel as papillomata have been demonstrated
in the laser plume. Good suction of smoke and laser operating masks are usually
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sufficient. Eye protection must be used to avoid laser damage to the globe.

Fig. 10. Postoperative view

Interferon is a product of human leukocytes, although it is now produced via
recombinant technology. Interferon treatment appears to slow the rate of growth
without curing the disease. Although some antiviral agents (egg, cidofovir, acyclovir)
also may slow the rate of regrowth of lesions, they are not curative. Eventually, somepatients may enter remission.
Side effects include flu-like symptoms, elevation of hepatic enzymes, renal
insufficiency, anorexia, seizures, GI distress, and transient numbness. There is somesuggestion that neutralizing antibodies may blunt its beneficial effect and that thismay vary by brand. Results may vary considerably from patient to patient.
The surgical interventions were performed under general endotracheal anaesthesia, concerned about the anesthesiological safety aspects of laser surgery. We
didn’t notice any accidents or complications regarding CO2 laser procedures during
these interventions. No intra and postoperative haemorrhages occurred.
We used a minimal postoperative aftercare: antibiotic prophylaxis for 5 days
with ampicilin 2g per day im to prevent a bronchopneumonia or a local infection;
mucolytics; minor antalgics in the first 24-48 hours to prevent local pain.
We have no postoperative complication. None of the 28 patients had impairment of voice. Clinical examination revealed regression of papillomas in all patients.
Functional results were very good at 24 patients (85.71%) and good at 4 patients(14.28%). The results of these 28 patients are:
• Free of disease in 19 cases, 67.85%• 9 patients (32.14%) had partial response to the combined treatment, inthese cases second intervention was needed. No perioperative or postoperativecomplications and no severe side-effects were noted.
Time interval from surgical intervention to recurrences was on the average 12months, between 3 and 28 months. In cases of recurrence, the patient underwent
CO2 laser removal of the papillomas formations and adjuvant therapy with IFN
alpha 2b.
Average hospitalisation was 5 days, between 3 days to 12 days for the patientwith bronchopulmonar complication. Treatment failures, recurrences are due to its
normal evolution of the disease.

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DISCUSSIONS
Since the introduction of the endoscopic CO2 laser microsurgery into clinicalotolaryngological procedures, laser surgery for RLP has been gaining increasing
importance. RLP represent the most common larynx benign tumour.
The typical course is of recurrent lesions requiring frequent debulking. However,every patient is different and treatment must be tailored to the individual. Somepeople require such frequent debulking that tracheotomy is necessary for airway
protection. This unfortunately frequently leads to lesions around the tracheotomy site(in essence, an iatrogenic squamociliary junction), but traditional thinking has beenthat this predisposes to distal tracheal disease. A recent study of children, however,suggests that those who require tracheotomy have more aggressive disease andhave distal disease prior to tracheotomy, and thus recommends that no child be
subjected to a tenuous airway merely to avoid tracheotomy (14). Tracheotomy has
two inconvenients: first it facilitates the spread of papillomas and second: produces
moisture changes which also facilitate the spread of the virus.
Interestingly, virus can be detected in the normal mucosa adjacent to lesions. It
is thought that this provides a reservoir for regeneration of new papillomata (15).
The natural history of the disease includes spontaneous remission. It is
impossible to know to whom or when this will occur. The causes of remission are
not known, and while the exact incidence is not known, it is thought that approximately one-third will remit by age forty. This makes clinical trials somewhat
difficult inasmuch as it confounds the ability to tell who remitted due to treatment
and who remitted due to the natural history of the illness.
A recent survey of otolaryngologists showed that 92% favoured the laser (16).
The KTP laser can be used for more distal disease.
Management of the airway is controversial. In the apnoea technique thepatient is intubated and administered 100% oxygen for a period of time. The tube
is then removed for a period of time while the surgeon works. The patient is thenreintubated and reoxygenated. This may be advantageous in paediatric airways inwhich there are not much room to work around a tube. Other methods include
use of a laser-safe tube and spontaneous ventilation. Another common method is
jet ventilation. Although this is generally felt to be safe, there is concern that thismethod may lead to distal inoculation of the virus. In patients with an existing
tracheotomy, a metal tracheotomy tube can be placed to allow laser surgery to
be carried out safely. A recent survey of otolaryngologists found the percent who
favoured the various techniques as follows: laser-safe tube 46%, jet ventilation
25%, apnoeic 16%, and spontaneous 12% (16).
Due to the nature of the disease adjunctive measures and alternative treatments have been sought out. In the absence of an effective antiviral agent, use ofdrugs that will augment host defence is a reasonable approach. Seventy-five to
80% of patients respond to interferon, with a complete response in about 30%.
Interferon does not eradicate the virus, and relapse may occur after discontinuationof treatment. Therapies which have been explored but rejected include steroids, estrogens, cryotherapy, cautery, ultrasound, radiation, vaccines, resin of podophyllum,transfer factor, levamisole, suction diathermy, lymphokines, escarotics, calandine,
magnesium, and antibiotics (17). Newer therapies which have been tested include
alfa-interferon, indole-3-carbinol, acyclovir, retinoic acid, ribavirin, methotrexate,
cidofovir, and photodynamic therapy. Case reports suggest that combined treatment
with acyclovir or retinoic acid may be beneficial in patients with recurrent diseaseduring interferon treatment.
Some studies and uncontrolled observation in patients with RRP indicate thata diet high in cruciferous vegetables (egg, cabbage, cauliflower, broccoli, Brussels
sprouts) may have a favourable effect. Researchers hypothesize that indole 3-carbinol is the active agent in these vegetables; its role is under study.
Fiziologia - Physiology  2009.19.2(62)Cidofovir (Forvade, Vistide) - Currently approved for treatment of CMV retinitis
in AIDS. Cidofovir is the first member of a group of antivirals known as acyclic phosphonate nucleotide analogs. In infected cells, nucleotide analogs such as cidofovir
inhibit viral DNA polymerase, which is responsible for replication of new viral RNA
and DNA. Because HPV is the causal agent for RRP, eradication of the virus offers
the potential for cure. Intralesional use is beneficial.
The etiologic link between maternal condyloma at delivery and JORRP in theinfant was first recognized by Hajek (2) in a case report in 1956. This observation
was supported by additional case reports (4, 18, 19). Subsequent virologic studies
fully substantiated the link between genital condylomas and JORRP. HPV types 6
and 11 which are responsible for 80-90% of the condylomas are documented innearly 100% of JORRP (20,21,22). Transmission of the virus from mother to infant
is believed to occur predominantly intrapartum (18). Cases of JORRP rarely give a
history of caesarean birth, an indication that caesarean delivery decreases the riskof acquiring JORRP. (23) Adult-onset RRP (AORRP) is also caused by infection with
HPV-6 and HPV-11 but very probably, the infection is not acquired at birth (24).
Genital tract infection with HPV-6 and HPV-11 is common, but JORRP is rare.
Data are not available to make a reliable estimate of the risk of transmission froman infected mother to a child but this risk is perceived to be low (23,24).
The guideline on perinatal care of the American College of Obstetricians
and Gynaecologists states that "caesarean delivery is not recommended solely
to protect the neonate from HPV infection". We have estimated that the risk oftransmission of JORRP from a condylomatous mother to an infant may be 1-3%,(25) and could be as high as 8% for first-born children of teenage mothers (Bishai
et al., unpublished data). The assumptions on which these estimates are made, as
well as other considerations (e.g., maternal morbidity due to caesarean delivery,
cost-benefit analysis of caesarean delivery), need to be debated before a populationwide policy regarding the prevention of JORRP by caesarean deliveries is instituted,
but personal choice is a different matter. It is highly probable that some womenat risk would be willing to personally incur the extra expense and operative risk of
caesarean delivery to eliminate a 1%-8% chance of JORRP in their child. Several
new approaches toward prevention and treatment of condylomas are promising(26,27). Any treatment that would reduce the HPV viral burden in the genital tract
during labour, or diminish fetal contact with maternal virus, would likely decrease
the incidence of JORRP (28,29).

CONCLUSIONSIn conclusion, RRP is a disease which causes a substantial human and financial
cost to the public. HPV has been shown to be the aetiology. It affects people of
all ages. Life-threatening airway obstruction may develop. The natural history ispoorly understood but is characterized by spontaneous remission in some patients.
Treatment is essentially palliative with surgical debulking. Various adjunctive drugs
have been developed which slow but do not eradicate the progression of disease insome patients. Currently there is ongoing research aimed at improving the treatmentof this insidious disease.
Because the disease is uncommon and requires direct laryngoscopy for
diagnosis, children usually have symptoms for a year before a physician makes thediagnosis. The morbidity of this disease has been studied more completely for JORRP,
in which the average number of surgical procedures required is 4.4 per child per year,
and the average number of procedures per child's lifetime is more than 20.
Although they can be found anywhere in the aerodigestive tract, there appears
to be a predilection for areas where there is a junction of squamous and cilliary
epithelium. This includes the limen vestibuli (junction of the nasal vestibule andthe nasal cavity proper), nasopharyngeal surface of the soft palate, midzone of
2009.19.2(62)  Fiziologia - Physiology

the laryngeal surface of the epiglottis, upper and lower margins of the ventricle,
undersurface of the vocal folds, and the carina and bronchial spurs.
Because the disease is rare, large-scale trials of medical therapies have notbeen possible; however, several agents are available that appear to increase theintervals at which surgical debulking is required. These include systemic andintralesional interferon, intralesional cidofovir, indole 3-carbinol, and photodynamic
therapy. Agents that demonstrate variable effects include cimetidine, acyclovir,
and retinoic acid.
The carbon dioxide laser is the preferred method for resection of papillomas
because it affords good haemostasis and minimizes potential thermal injury ofsurrounding healthy tissues.
Combined treatment with Endoscope CO2 laser Microsurgery and Interferonalpha 2 b is considered to be the election method in RLP, offering long remission
periods and sometimes healing. CO2 laser ablation permits the protection of vocal
cords, allowing a normal breathing, and especially a normal voice. The associatedtreatment with IFN alpha 2b has largely improved the prognosis of this viral disease.
The good functional results, with the local control of the tumour for a long periodof time and a short hospitalization represent the most important advantages ofthis treatment method.
This combined treatment is a method of choice in laryngeal papillomatosis inchildren, causing longer remission of the disease. Surgical ablation with laser CO2
is the elective treatment and its combination with IFN improved the prognosis of
this recurrent viral disease. Reachable functional results, good tumour control, short
hospitalization makes it a favourable treatment in paediatric patients.
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MANAGEMENTUL PAPILOMATOZEI LARINGIENE
RezumatObiective: Papilomatoza laringiană juvenilă (PLJ) este o afecţiune de o etiologie virală cauzată de Virusul Papilomatozei Umane tip 6 şi 11, cu o incidenţă de
4-7 cazuri/1 milion, caracterizată prin multiple recidive ale formaţiunilor tumorale benigne ale mucoasei laringiene, care determină o morbiditate semnificativă
în patologia pediatrică ORL. Reprezintă cea mai frecventă tumoră benignă a laringelui, cu o morbiditate ridicată, dificil de tratat datorită recidivelor şi adiseminării de-a lungul tractului respirator. Scopul studiului este de a evalua rezultatele tratamentului combinat prin microchirurgie endoscopică cu laser
CO2 şi Interferon alpha 2b la copiii cu papilomatoză laringiană.
Material şi Metodă: In acest studiu au fost incluşi, în perioada 1996-2008, 28 de pacienţi cu vârste cuprinse între 2,9-9 ani diagnosticaţi cu papilomatoză
laringiană. Tratamentul a constat în microchirurgie endoscopică cu laser CO2 (excizia şi vaporizarea formaţiunilor tumorale), urmată de tratament adjuvant
cu Interferon alpha 2b. Traheotomia nu s-a practicat la nici un caz.
Rezultate: Examinările endoscopice postoperatorii au constatat regresia formaţiunilor papilomatoase la toţi pacienţii. La 19 pacienţi (67,85%) s-a constatat
regresia completă a afecţiunii, după microchirurgia cu laser CO2, ca metodă primară, şi tratamentul adjuvant cu Interferon. Răspunsul parţial la tratamentul
combinat s-a constatat la 9 pacienţi (32,15%). In aceste cazuri a fost necesară reintervenţia. Nu s-au înregistrat complicaţii intraoperatorii şi postoperatorii,
precum şi efecte adverse severe ale tratamentului cu Interferon.
Concluzii: Tratamentul combinat al papilomatozei laringiene reprezintă terapia de elecţie cu perioade de remisiune lungi. Acest tratament a îmbunătăţit
prognosticul acestei afecţiuni virale. Rezultatele funcţionale bune, cu controlul local al formaţiunilor tumorale papilomatoase, spitalizarea de scurtă durată,reprezintă elementele favorabile ale acestei metode de tratament la copiii cu papilomatoză laringiană.
Cuvinte cheie: papilomatoză laringiană juvenilă, tratament cu laser CO2, tratament cu IFN alpha 2B

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Fiziologia - Physiology  2009.19.2(62)URBAN NOISE AND ENVIRONMENTAL COMPLAINTSIN TIMISOARA
ERNEST PUTNOKY
Ministry of Environment – IB Timisoara

ABSTRACT
Urban road noise has always been an important environmental problem for man from ancient history. But despite the fact proven by measurements andcalculations that road traffic is the main source of noise in urban areas, citizens have the tendency to accept the situation as part of modern way of life and as
a “necessary evil”. This paper compares the results of noise measurements and the noise related complaints of the citizens from Timisoara.
Keywords: urban noise, noise maps, complaints, environmental protection

INTRODUCTION

MATERIALS AND METHODS

Noise has always been an important environmental problem for man. In ancientRome, rules existed as to the noise emitted from the ironed wheels of wagons whichbattered the stones on the pavement, causing disruption of sleep and annoyanceto the Romans. In Medieval Europe, horse carriages and horse back riding were notallowed during night time in certain cities to ensure a peaceful sleep for the inhabitants. However, the noise problems of the past are incomparable with those of modern
society. An immense number of cars regularly cross our cities and the countryside.
There are heavily laden Lorries with diesel engines, badly silenced both for engine
and exhaust noise, in cities and on highways day and night. Aircraft and trains add tothe environmental noise scenario. In industry, machinery emits high noise levels andamusement centers and pleasure vehicles distract leisure time relaxation.
In comparison to other pollutants, the control of environmental noise has beenhampered by insufficient knowledge of its effects on humans and of dose-response
relationships as well as a lack of defined criteria. While it has been suggested thatnoise pollution is primarily a "luxury" problem for developed countries, one cannot
ignore that the exposure is often higher in developing countries, due to bad planningand poor construction of buildings. The effects of the noise are just as widespreadand the long term consequences for health are the same. In this perspective, practicalaction to limit and control the exposure to environmental noise are essential. Suchaction must be based upon proper scientific evaluation of available data on effects,
and particularly dose-response relationships. The basis for this is the process of risk
assessment and risk management.
The extent of the noise problem is large. In the European Union countries about 40%of the population is exposed to road traffic noise with an equivalent sound pressure levelexceeding 55 dBA daytime and 20% are exposed to levels exceeding 65 dBA. Taking all
exposure to transportation noise together about half of the European Union citizens are
estimated to live in zones which do not ensure acoustical comfort to residents. More than
30% are exposed at night to equivalent sound pressure levels exceeding 55 dBA which
are disturbing to sleep. The noise pollution problem is also severe in cities of developing
countries and caused mainly by traffic. Data collected alongside densely traveled roads
were found to have equivalent sound pressure levels for 24 hours of 75 to 80 dBA (2).
For these reasons, in this paper, the noise level measurement are put side by sidealong the Timisoara City Hall’s noise maps and the peoples response to noise issues
recorded as written complaints to the environmental authorities of the city of Timisoara

During 2001 and 2006 a number of 630 daytime and nighttime measurements
were conducted in randomly selected measurement points in the urban area ofTimisoara by the environmental authority of Timisoara. The measurement points wereclassified according to the function of the selected urban area in traffic, residential,
recreational, protected (medical treatment & care) and industrial areas (5).
The measurements were made with a Bruel&Kjaer 2238 Mediator (Class 1 Integrating Sound Level Meter with logging software) fitted with an outdoor kit (free-fieldmicrophone with windscreen, cable preamplifier extension and tripod).
The microphone was set at a height of 1.3 m form the ground level, accordingto Romanian noise measurement standards (5).
Traffic area measurements were made at the edge of the pathway (25m fromthe axis in the railways case), industrial and protected area measurements were madealong the specific perimeter, recreational area measurements were made inside thearea, as central as possible and in the residential area measurement were conducted at
a distance of 3m form the façade of the buildings, facing the noise sources, accordingto Romanian measurement standards (4).
Measurement data included LAeq (A-weighted equivalent noise level) L90(background noise) and L10 (maximum noise). Lowest sound pressure levels andpeak levels were also recorded. The measurement unit was the dBA (A-weighted
decibel) (4).
Computed Data in the process of completing the first stage of city’s noise mappingby the Timisoara City Hall according to European Commission 2002/49/EC Noise Directive were used to asses the percentage of population affected by various type of noise:road traffic, public rail transportation, industrial and air traffic noise (1,7).
Last but not least, a number of 1523 written complaints addressed during
2001-2006 to the environmental authority were processed and those referring toenvironmental noise issues were studied in order to find possible links between the data
provided by noise measurements, noise maps predictions and citizen’s opinions.

RESULTS
Daytime and nighttime measurements of the A-weighted equivalent noise levelindicated that the main cause of the urban noise is the road traffic. This type of noise
source is combined with the public rail transportation traffic (trams and trains).

Received March 2009. Accepted April 2009. Address for correspondence: Ernest Putnoky, Ministry of Environment – IB Timisoara, Romania

2009.19.2(62)  Fiziologia - Physiology

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No matter of the type of the zone in which the measurements took place (residential, recreational, industrial or protected) the overall noise level was more or lessaccurate defined by the technical category of the neighboring roads (6): 1st categoryroads at the city limits with measured LAeq between 75 dBA and 80 dBA, 2nd categoryroads – the city’s main boulevards and roads with measured LAeq between 60 dBA
and 74 dBA, 3rd and 4th category roads – connecting street between main roads, usually with less traffic and lower speed limits (3) with measured LAeq between 49 dBA
and 59 dBA. The majority of roads cross the residential area of the town so the impact
upon the population of road traffic generated noise is significant.
According to Timisoara City Hall noise mapping, completed in 2008 (7), 52.8%of the population from Timisoara’s residential area is affected by daytime road traffic
noise levels over 65 dBA; 15.0% of the population is exposed to noise levels exceeding 75 dBA.
The above values should be compared with the 5.5% of the population exposedto tram and railway generated noise (in the 55 dBA to 65 dBA domain) and 0.0% of
the population exposed to air traffic generated noise.
Industrial activities exposes about 0.6% of the population to noise levels between
55 dbA and 65 dBA.
The overwhelming impact of road traffic on noise level in the residential area
is evident.
These numbers are calculated projections of the Lden which is an indicator of
the overall noise level during the day, evening and night which is used to describe
the annoyance caused by exposure to noise, according to EC noise mapping specifications (1).
As for the citizens’ complaints regarding different environmental issues, thestudy of the 2001-2006 archives of the Environmental Authority in Timisoara led to
the following results:
from the total number of written complaints (1523), 31.5% are referring
to environmental noise;
the rest of the complaints refer to air pollution (44.1% - smoke, dust,gas, smell) and water and soil pollution (24.4% - dejections, waste, oil and derivates,
pesticides and chemicals/poisons)
The noise complaints are divided as:
35.2% are reporting daytime annoyance from noise resulted in the activity of small and medium enterprises (car services, car washes, metal works, bakeries,
warehouses, construction yards, saw-mills, factories);
33.3% are reporting nighttime annoyance from high level music played
inside neighboring bars and restaurants;
10.8% are reporting nighttime annoyance from high level music played
inside neighboring clubs and discotheques;
5.4% are reporting nighttime annoyance from noise generated by air
conditioners and chillers;
4.5% are reporting daytime and nighttime annoyance from noise generated by football supporters, football matches and celebrations held in/around the city’s

main football arena;
8.1% are reporting daytime and nighttime noise from various sources(road/water/sewer/gas maintenance work, dogs, background noises like electrictransformer buzzing or pipes hissing, dogs barking, peddlers and illegal motorcycle
racings);
2.7% are reporting daytime and nighttime noise generated by the roadtraffic, including public rail transportation (trams and trains).

CONCLUSIONS
Measurements conducted in hundreds of randomly selected points of the urban
area of Timisoara indicate the road traffic as the main cause of the urban noise. Public
rail transportation (trams and trains) although affects a much smaller residential area
and only for short periods of time, generate high levels of noise that could result inserious annoyance especially during night time.
Noise maps calculated values are confirmed by field measurements and indicate
a semnificative impact on more than half of the city’s population.
Despite these affirmations and despite all the experimental data, only a small
percentage of the population reports road traffic to the authorities as being a serious
annoyance.
This attitude is due to several factors: the increase in demographic density, theincrease in the number of per capita vehicles, the fact that society is getting used tohigher noise levels and accepts road traffic as a “necessary evil”, and the widespread
ignorance regarding the consequences of noise as well as its remedies.
However, it is interesting to remark that citizens that dwell in residential areas
exposed to heavy daytime and nighttime road traffic have a much lower “noise acceptance threshold”and will promptly forward complaints for any newly arrived noise
source in the residential area, such as a loud music playing restaurant, but will continue
to accept or ignore the main noise generator: the road traffic.
REFERENCES
1. The European Parliament and Council: Directive 2002/49/EC - Assessment and Management of Environmental Noise, Bruxelles 2002
2. World Health Organization, Regional Office For Europe European
Centre For Environment And Health: Noise Guidelines For Europe,
1999
3. Drăgănescu G: Vibraţii şi zgomote, Ed. Politehnica, Timişoara,
2000
4. STAS 10009-88: Acustica urbană. Limite admisibile ale nivelului dezgomot urban.
5. STAS 6161/3-82: Acustica urbană. Metode de determinare a niveluluide zgomot în localităţile urbane.
6. STAS 10144/3-91: Elemente geometrice ale străzilor. Prescripţii
de proiectare.
7. Primaria Timisoara: Harta de zgomot a municipiului Timisoara, 2008
http://www.primariatm.ro/index.php?meniuId=2&viewCat=1314

ZGOMOTUL URBAN SI SESIZARILE LEGATE DE PROTECTIA MEDIULUIIN MUNICIPIUL TIMISOARA
REZUMAT
Zgomotul rutier urban a fost o problema de mediu inca din antichitate. In pofida faptului ca masuratorile si calculele indica traficul rutier
ca sursa principala a zgomotului urban, cetatenii au tendinta sa accepte situatia ca parte a modului de viata modern si zgomotul generat
de traficul rutier ca pe un “rau necesar”. Acest articol compara rezultatele masuratorilor de zgomot cu sesizarile legate de zgomot facute decetatenii orasului Timisoara.
Cuvinte cheie: zgomot urban, harti de zgomot, sesizari, protectia mediului
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Fiziologia - Physiology  2009.19.2(62)BOOK REVIEWSTRESSOLOGY, ADAPTOLOGY AND MENTAL HEALTHSORIN RIGA and DAN RIGA
Cartea Universitara Publishing, Bucharest, 2008, XII + 266 pages
Stressology, Adaptology and Mental Health”, published by Cartea Universitara Press, Bucharest, 2008, was written by Sorin Riga, MD, PhD, DHc, andDan Riga, MD, PhD, DHc, Directors of the Department of Stress Research andProphylaxis, from “Al Obregia” Clinical Hospital of Psychiatry, Bucharest. It isa monograph of excelence in a growing field of great interest, based on theauthors experience and work in the stress and anti-aging medicine.
The first part, “Stressology” starts with presenting Hans Selye’s concepts
about stress, adaptation and health. From a physiological and adaptive perspective, stress can be defined as the aggression by the environment on the human body, together with the specific adaptation mechanisms and nonspecific,
neuro-endocrine ones, leading to increase the individual’s resistance and toprevent morphological and physiological abnormalities, which may generate
pathological states. Under normal circumstances the psycho-neuro-endocrine
systems permit the individual to react appropriately to stressors, allowing
the adaptation to the conditions induced by the stressors. From a global andcomprehensive perspective, stress encompasses the socio-psycho-medical
dimension regarding the impact of stress in society, the relationship mental
health – stress, the ontogenetic dimension and the field of prevention/therapy. The book presents relevant data regarding the international progressin stressology, as well as aspects of the advanced Romanian researches, bothin stress and anti-aging medicine. The authors scientific contribution in thearea of psychopharmacotherapy of stress and adaptation is impressive and wellrecognized internationally. The recent brain researches - at macromolecular,
metabolic, subcellular, cellular and tissual levels, through advanced neuroscientific techniques – are the background for the concepts, innovations andanti-stress therapies, as well as of improved adaptation and longevity, areas
well presented by the authors. The phenomenon of stress is evaluated fromvarious perspectives: the stressors, stress responses, the psychiatric and psychosomatic stress-dependent disorders. Numerous scales, questionnaires andclinical algorithms, useful in stress research, are discussed in stress diagnosis
and evaluation, in both medical practice and in health systems services. Acuteand chronic stress-related disorders are connected with clinical descriptions
and guidelines of international classifications (ICD-10, WHO, Geneva, 1992
and DSM-IV-TR, APA, Washington, DC, 2000). In the end of this section, theauthors analyze psychosomatic disorders and medicine.
The second part of the monograph, “Adaptology” reviews the binomial relationship distress – eustress, homeostatic concepts, and adaptation
mechanisms, to formulate growth strategies of the bio-psycho-social resistance and the individual’s capability to adapt. It also presents allostasis, vitality
and vulnerability of human being. The General Adaptation Syndrome and its
pathology, the individual’s ability to cope (coping/anti-stress mechanisms),
the behavior with its typology and abnormalities are aspects approached withthe most recent information in the field, filtered through the authors’ vast
knowledge and experience. Their original contribution, the entropic cascade(distress – impairment – aging – polypathology) is the foundation for a2009.19.2(62)  Fiziologia - Physiology

new paradigm in medicine – both prophylactic and therapeutic, leading to
new perspectives in lifestyle, health and longevity management. The authors
have valuable researches and results in oxidative stress, diseases of the free
radicals, senescence: anti-stress, anti-impairment and anti-aging therapy(Antagonic-Stress® ).
Last part, “Mental Health”, presents aspects of mental health policies,
their institutionalization and organization at national and international levels,
through specialized structures. The concept of mental heath is defined andanalyzed in an integrative and dynamic vision, as part of the “health-mental
health” binomial, as expression of the quality of individual life and quality ofthe society at economic, administrative and social planes. In the chapter on
health and longevity management, the authors offer solutions, elaborating
pro-health and pro-longevity strategies, ways for the individual to optimize
his/her relationship with himself/herself and with the outside world (physical and psychical activation and rebuilding, emotional-cognitive-volitivebehavioral therapy, learning healthy lifestyles, anti-stress and anti-aging
programmes).
The book is a premiere performance in Romanian medicine, a reference
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work in the field, from theoretical, fundamental, experimental to appliedclinical perspectives and medical sociology. In a concise, impressive and very
well structured presentation, with an actual bibliography for each chapter, theauthors offer new concepts and strategies, modern studies in depth, of greatuse in teaching and research, as well as numerous diagnostic and therapeutic

tools, useful in medical practice. Therefore, drs. Riga’s contribution represents
a progress in the future medicine and personalized health. This monograph is
an asset for physicians, psychologists, psychotherapists, sociologists, specialists
in mental and public health, for all those interested and involved in promoting
healthy life patterns.

Assoc. Prof. Dr. Daniela Motoc

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Fiziologia - Physiology  2009.19.2(62)