Official Journal of the Romanian Society of Physiological Sciences

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CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
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Fiziologia - Physiology 2010supplement
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Official Journal of the Romanian Society of Physiological Sciences

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Fiziologia- Physiology 2010.20.2(66)
Fiziologia- Physiology 2010supplement

CONTENTS
1. In Memoriam Ioan Haulica.................................................................................................................................................................................................................................... 4
2. Health Systems in the European Union. An Overview............................................................................................................................................................................................ 5
Cristian Busoi
3. Contemporary Man in His Bio-Psycho-Socio-Ecological Dimension....................................................................................................................................................................... 8
Sorin Riga, Dan Riga, Aurel Ardelean, Francisc Schneider
4. REVIEW: Infections Associated with Pregnancy and Childbirth........................................................................................................................................................................... 11
Camelia Budisan, Constantin Ilie
5. C Reactive Protein in Diabetes Mellitus Type 2 Complicated with Diabetic Nephropathy..................................................................................................................................... 15
Matei D, Moldovan C, Muresan M, Micle L, Micle O, Burta O, Oros R, Muresan A
6. Autoimmune Endocrine Associations in Adult Patients with Affected Glycemic Metabolism and Thyroid Disorders............................................................................................ 20
Adriana Gherbon, Romulus Timar, Ioana Zosin
7. Long-term implications of cardiovascular and respiratory medications in elderly asthmatics............................................................................................................................. 26
Simona Macesanu, Elena Ardelean

8. Stromal Cells - Tumor Microenvironment Interactions – Part II........................................................................................................................................................................... 31
Florina Bojin, Calin Tatu, Oana Gavriliuc, Alexandra Gruia, Gabriela Tanasie, Carmen Tatu, Simona Anghel, Mirabela Cristea, Felicia Ciuculescu, Alexandru Tocut, Daniela Crisnic, Adrian
Carabineanu, Daciana Nistor, Carmen Bunu, Virgil Paunescu
9. The Analysis of State of Tension and Strain at the Level of Cervical Vertebrae (Atlas) Through Finite Element Method. ........................................................................................................... 37
Maciovan Adina
10. Serological Immunopathology of Patients with Myasthenia Gravis................................................................................................................................................................... 42
Bogdan Istrate, Crisanda VîlciuCUPRINS
1. In Memoriam Ioan Haulica.................................................................................................................................................................................................................................... 4
2. Sisteme de sanatate in Uniunea Europeana. Prezentare de ansamblu................................................................................................................................................................... 5
Cristian Busoi
3. Omul contemporan in dimensiunea sa bio-psiho-socio-ecologica........................................................................................................................................................................ 8
Sorin Riga, Dan Riga, Aurel Ardelean, Francisc Schneider
4. REVIEW: Infectii asociate cu sarcina si nasterea................................................................................................................................................................................................... 11
Camelia Budisan, Constantin Ilie
5. Proteina C reactiva in diabetul zaharat tip 2 complicat cu nefropatie diabetica................................................................................................................................................... 15
Matei D, Moldovan C, Muresan M, Micle L, Micle O, Burta O, Oros R, Muresan A
6. Asocieri endocrine autoimune intalnite la pacientii adulti cu alterari ale echilibrului glicemic si afectiuni tiroidiene.......................................................................................... 20
Adriana Gherbon, Romulus Timar, Ioana Zosin
7. Implicatiile pe termen lung ale medicatiei cardiovasculare si respiratorii la varstnicii astmatici.......................................................................................................................... 26
Simona Macesanu, Elena Ardelean
8. Interactiunea dintre micromediul tumoral si compartimentul stromal – Partea II.............................................................................................................................................. 31
Florina Bojin, Calin Tatu, Oana Gavriliuc, Alexandra Gruia, Gabriela Tanasie, Carmen Tatu, Simona Anghel, Mirabela Cristea, Felicia Ciuculescu, Alexandru Tocut, Daniela Crisnic, Adrian
Carabineanu, Daciana Nistor, Carmen Bunu, Virgil Paunescu
9. Analiza starii de tensiune si deformatie la nivelul vertebrei cervicale atlas cu ajutorul metodei elementului finit.............................................................................................. 37
Maciovan Adina
10. Patologia imunoserologica a pacientilor cu Miastenia Gravis............................................................................................................................................................................ 42
Bogdan Istrate, Crisanda Vîlciu

2010.20.2(66)  Fiziologia - Physiology3

IN MEMORIAM IOAN HAULICA

He was born October 29, 1924, in the village of Ipatele, from thecounty of Iasi, in a numerous family and attended high school in Iasi.
After graduating from the Faculty of Medicine in 1949, he worked forthree years as a junior assistant in the departments of Pharmacology andPathophysiology of the Institute of Medicine and Pharmacy Iasi. Being
dismissed, he moved to the Institute of Normal and Pathological Physiology of the Romanian Academy in Bucharest, where he worked for 12
years as a researcher under the guidance of academics D. Danielopolu
and Grigore Benetato.
After obtaining a doctoral degree in medical science in the fieldof physiology, he returned to Iasi in 1964, when he became associate professor of physiology in the same department of the Institute ofMedicine in Iasi.
After obtaining the title of docent and obtaining the position of
professor in 1968, he took over the coordination of the scientific andteaching activity of the department, where he continued working until his
retirement in 1994.
Received training courses in USA, Austria, Poland, USSR, Germany,and France he worked as a “visiting professor” at University Village Connecticut (U.S) during the school year 1969-1970.
In 1972 he was able to organize in Iasi an international workshop of“Synaptic Transmission” for a period of three weeks, sponsored by IBRO/UNESCO and attended by professors from the U.S. and 12 European
countries.
During his half of a century of relentless pursuit of the secrets of
the human body he made significant contributions to important fieldsof physiology and human biology among which are worthy of mention
a wide range of topics of normal and pathological physiology, making
original contributions to some of the issues under study. He carried out
fundamental research on the physiology and pharmacology of autonomic organs, functional biochemistry of brain, extrarenal origin reninangiotensin system, neuroendocrinology of acute and chronic stress,
neurohumoral bases of pain and stress analgesia, functional implicationsof free radicals and endogenous antioxidants, role of endothelium andnitric oxide in vascular reactivity, etc. Much of the experimental researchwere confirmed and quoted by various authors in the country and abroad.
The discovery of the intrinsic renin-angiotensin system in the pituitary andpineal glands in mammals and humans and the identification of the roleof the purinergic nerve fibers from the myenteric plexus, spinal ganglia,
spinal cord, brainstem, cerebellum and hypothalamus are internationallyrecognized contributions.
In collaboration with the “Max Delbrück” Center for Molecular
Medicine in Berlin he discovered a new active angiotensin II-forming
enzyme in the human brain and endocranial glands (pituitary, pineal)called chimase, which predominates over the angiotensin converting
enzyme in the brain.
He also brought cognitive contributions to the study of the heartand vascular renin-angiotensin system, eye, uterus and ovary, and theneuro-humoral basis of pain and the role of free radicals in the central
neurochemical imbalances caused by stress. During the last years, hegave a special attention to the role of vascular endothelial reactivity,
obtaining experimental evidence for the involvement of nitric oxide (NO)both as endothelial relaxing factor as well as neural messenger involvedin nociceptive sensitivity.
He has published over 300 papers in specialized journals in the
country and abroad. He elaborated and published as first author or incollaboration three treatises of human physiology and eight monographs,
out of which three received the Romanian Academy Award (“Autonomic

4

Nervous System” in 1975, “Local Hormones” in 1983 and “Synaptic
Transmission” in 1999).
He was author or coauthor of 10 patents. He participated in numerous scientific events in the country and abroad, reports, communications, posters and roundtables. He was Dean of the Faculty of Medicine(1972-1976) and pro-rector of the Institute of Medicine and Pharmacy
Iasi (1981-1988).
He was a member of several Romanian and international scientificsocieties: International Society of Physiological Sciences, FEPS, Societyof Physicians and Natural Scientists from Iasi, International Society for the
Study of Brain (IBRO), European Society for Pineal Studies, European
Federation of Biological Sciences, Society for Neuroscience, Psychoneuroendocrinology Society and others.
He was deputy chairman of the Academy of Medical Sciences andpresident of the Society of Physiological Sciences in our country until 2004.
He was president of the Society of Physicians and Natural Scientists from
Iasi between the years 1990-1994. He was elected Fellow of the RomanianAcademy in December 1991 and he became a full member in 1994.
In 1999 he was granted the title of Doctor Honoris Causa by the
University of Medicine and Pharmacy, Tg. Mures and in 2003 by the
University “Ovidius”, Constanta. In 2002 he received the Order “Star of
Romania” in the rank of Knight.
In 1997, three years after his retirement, he founded the Laboratoryof Experimental and Applied Physiology subordinate to the Iasi branchof the Romanian Academy where he continued to pursue research on
the renin-angiotensin system, biological properties of deuterium depleted
water the influence of radical species on vascular reactivity and central
nervous activity.
Until the very moment of his death, Professor Ioan Haulica continued
to study and research as honorary head of the Laboratory of Experimental
and Applied Physiology, to write and try to impart to the others out of therichness of his knowledge and experience. His last book, a treaty on theautonomic nervous system was finalized mere months before his untimely
death, thus denying him the joy of seeing it published.
“The Professor” will remain forever in the memory of thousands offormer students, PhD students, colleagues, collaborators and friends,
upon whose lives he would have left an ineffaceable impression.
		Physiology Department, UMF Iasi
Prof. Simona Slatineanu, MD, PhD

Fiziologia - Physiology  2010.20.2 (66)HEALTH SYSTEMS IN THE EUROPEAN UNION.
AN OVERVIEWCRISTIAN BUSOI
Public Health Department, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACTAn analysis of the existing healthcare systems of the EU Member states shows a significant degree of diversity while all were inspired by the two major healthcare system models known as the Beveridge and the Bismarck models. The EU healthcare systems
vary according to the financing of the system, to the proportion of the private healthcare sector in the overall system, the coverageof the insurance package, the more or less freedom to choose the service provider, the presence of guidance mechanisms towards
a certain type of care etc. The Romanian healthcare system has suffered a series of transformations since 1989, a process that isstill ongoing and which is to be related to the struggle for democratization and modernisation of Romania society.
Key words: healthcare system, EU.

MATERIAL AND STRUCTURE OF HEALTHCARESYSTEMS
Beveridge system
With regard to the structure of healthcare systems, there
are two worldwide known models that have become the sourceof inspiration for the majority of the European national healthcare systems.
One of them is the so called Beveridge system which
was adopted by some EU Member States, namely the United
Kingdom and the Kingdom of Sweden. It was named after its
founder, Lord Beveridge and it is a healthcare system financedby the general tax system, where the entire population is granted
access to healthcare services. The total amount of payments towhich the patient is entitled is not linked in any way to the amountof the wages earned and health care services are offered toeverybody without any formal obligation of prior contribution to
the healthcare system. The responsibility for the management of
Beveridge healthcare systems lies with a professional administration under parliamentary scrutiny. Member States such as Italy,
Spain, Greece and Portugal have been more or less influenced
by the British model.
Bismarck model
The second important social protection model which is tobe found in Germany as well as in the Benelux countries is the
Bismarck model, which also bares the name of its founder, theformer Prussian Chancellor, Otto von Bismarck. The insurance
contributions are established as a percentage of the salaries andare managed by independent health insurance funds which are
to be chosen by each person. Thus, unlike the Beveridge system, the Bismarck system is not controlled by the state and theactivity and management of the contributions by the healthcare

insurance funds is not at all scrutinized by the parliament. The
healthcare services package to which each insured person isentitled is established in the framework contract which is negotiated by medical practitioners and healthcare insurance funds
representing the interests of the insured patients. Moreover, thecompulsory social insurance system is designed to cover people
depending on their membership of a socio-professional group.
One of the most representative healthcare systems within the
EU is the French one, which is characterized by the universalityof access to medical care, the freedom of medical practitioners
and the free choice of the provider as well as by the fact that the
management of the social security is done by social partners.
The system depends on the monopoly power that insurance
funds have on users as well as on the monopoly of the professional trade unions which are the only authorized to represent
the insured within the insurance funds boards.
An original case is the Italian one due to its hybrid character
combining the Anglo-Saxon model of healthcare system organization with the partial financing by patients. As in Germany
and France, the Italian social protection system has developed
through a plurality of professional regimes.
When it comes to choosing a healthcare system model,
one ought to take into account the role of the state through its
different agencies. At this point, the so-called ideological factor
comes into play as those political parties which are generally
supportive of an active role of the state in economy and thesociety level on a larger scale, will also be the supporters ofstate-controlled healthcare systems and vice versa. For the time
being, there is no entirely privatized or nationalized healthcare
system in the European Union. The most frequent situation iswhere state-controlled healthcare systems borrow some of thefeatures of healthcare systems based on marked mechanisms,
and the other way round. This happens because there is a risk

Received May 2010. Accepted June 2010. Address for correspondence: Busoi Cristian, MD, Public Health Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, 2A Eftimie Murgu
Square, Timisoara 300041, Romania, e-mail: cristian.busoi@gmail.com

2010.20.2(66)  Fiziologia - Physiology

5

of market failures and healthcare is a sector where the stateintervenes in order to correct such failures and to make sure thatthe population has broad has an as broad as possible accessto medical care. Therefore, the principles of state control andmarket mechanisms may be complementary (2). This is the idea
lying behind the classification made by some authors who referto three large groups of healthcare systems: liberal systems,
nationalized systems and intermediary systems.
IN-PATIENTS CARE
Regarding in-patient care, a high degree of diversity canbe noticed within the EU with a varying weight of the public
and private sectors within healthcare systems. Thus the role ofthe private sector varies from maximum (in the Netherlands) tominimum. In the French system, for instance, 63% of the total
amount of hospital beds belongs to the public sector, whereas
only the remaining 37% have private owners. On the other hand,out-patient medical care in France is provided by medical doctors
who work in private practices. There are also other cases thatdeserve full attention, namely the case of Belgium and Germany,
where the private sector covers more than 50% of the overall
healthcare system, even if this figure is slightly lower that in the
Netherlands. Denmark and Sweden are on the opposite side,with an almost absent private sector (1).
As a general rule, in countries with intermediary healthcare
systems, out-of-pocket payments for medical care are lower thenin those with liberal healthcare systems and higher then in thosecountries that have chosen a nationalized system. France andGermany are good illustrations of this, as fees raise from 74%
and 64% respectively.
FINANCIAL ISSUES IN EU MEMBER STATES
If we study carefully the healthcare system of EU Member
States, diversity can be noticed, not only at the organizational
level but also at a financial one.
Countries finance their healthcare system through taxes orsocial insurance contributions, or even a combination of the two.
The system based on budgetary resources which come fromcontributions is highly used in Great Britain, Ireland, Denmark,Finland, Spain and Portugal. Other countries like Greece and Italy
have chosen a mixed system where a part of the resources comefrom contributions and the rest come from the social healthcare
insurance budget. Also, countries such as Belgium, Germany,
Luxembourg and Austria have expressed their preference fora funding predominately based on social healthcare insurance
contributions. One example is Luxembourg where 60% arefrom social healthcare insurance fees and the rest of 40% of
the expenses for healthcare are financed by state. The extreme
examples are France and the Netherlands which have systems
that are exclusively financed by the social insurance budget, towhich the population give their contribution.
Countries which have a system financed by the contributions to social healthcare insurance budget have different social
security schemes. In some countries, the citizens have thefreedom of choosing their insurance provider, having at their
6

disposal many social healthcare insurances funds, such a way
is found in Germany, Belgium and the Netherlands. In othercountries like France, Austria or Luxembourg, the citizens haveno saying in the matter, affiliation being made on the basis of
their professional activity.
In countries where there is a national healthcare system, thepopulation is regularly covered by that insurance, in comparison
with countries which have chosen a social insurance system (on acontribution basis) like France, Austria, Luxembourg or Belgium,
where self-employed and employees are required to take an insurance, and thus to contribute to the healthcare insurance fund.
In the Belgian case, self-employed are only required to choose
the state healthcare insurance system for hospital care, while forprimary care or secondary care, they may as well opt for private
health insurance. The French system is also an interesting casewith a basic insurance system which doesn’t cover the wholetreatment, but which is complemented by a system of co-paymentthat may be covered by supplementary insurance (“mutuelle”).
Another element that deserves attention is the total exclusion of
the complementary insurance scheme of people with very highincomes, as it is the case in the healthcare system of Germanyor the Netherlands. The difference between the two cases isthat in Germany, these groups of citizens have the obligation
to contribute to a private healthcare system, in comparison with
the Netherland where the signing of this kind of insurance is left
at the judgment of the person concerned, with no obligation likethe case of Germany.
After a detailed analysis of healthcare systems structure in
the EU, we are able to conclude that EU healthcare systems havea certain number of features in common, despite their obvious
diversity. Under the institutional and financing aspect, thesecountries may be grouped in six different categories identified in
an OCED report, which proved to be extremely pertinent, empirically speaking (1). The first group is composed by countries which
rely mainly on market mechanisms regarding both the insurance
system as the provision of medical care services (this is the caseespecially in Germany, the Netherlands and Slovakia). Two othergroups are relatively close because of their public basic insurance
system combined with offering medical services based on market
mechanisms. These two groups differentiate themselves throughthe mechanisms of guiding the patient towards a certain type of
medical care (primary, secondary or tertiary without a large freedom of choice for the patient), as well as through the existence,
more or less pronounced, of private health insurance schemes
as a complement to basic insurance (in the first group we have
Belgium and France, while in the second, we find Austria,
Greece, Luxembourg or the Czech Republic). The fourth groupof countries, of which Sweden stands out as most representative,is the one in which the patient has a very large freedom of choice
and where there is no patient guidance mechanism towards acertain type of medical care, but in which private health services
represent a very low proportion. The last two groups are thosewhich are composed by public healthcare systems extremely well
regulated, but which differentiate themselves through the levelof constraint exerted by patient guidance mechanisms towards
Fiziologia - Physiology  2010.20.2 (66)different types of medical care and also through the intensity ofbudgetary constraint in the healthcare sector. In the first of these
two groups we may find Denmark, Finland, Portugal and Spain,
whereas in the second and last group, countries such as Italy,
Ireland, Great Britain, but also Hungary and Poland from the
new Member States of the EU.
ROMANIAN HEALTHCARE SYSTEM
Romania has operated major reforms after December 1989.
The communist system, of a totalitarian and statist type, wasreplaced by institutions of a democratic and pluralistic society.
Right after the Revolution the main democratic institutions, theParliament and political parties, were built and gradually all theinstitutions of the Romanian society suffered changes throughthe modernization and democratization process.
The transformations in the healthcare sector were not lesssignificant. The healthcare system in the communist period,
under-financed, statist and centralized was replaced with a healthinsurance system, which combines some of the free market
mechanisms, a sector of public hospitals currently in a processof decentralization, as well as private initiatives in out-patient of
in-patient care systems. The transformation was yet slow andfilled with hesitations, uncertainties and controversies.

With all the changes produced, some influences of the communist period have still remained - the illusion of total freenessof medical services, a modest budget given to the healthcare
system which led to a chronic under-financing, a lot of the aspects in hospital system structure, the relations within hospitals
especially those in university centers, the patient-doctor relation
or informal payments.
Despite these influences and reminiscences, the system
underwent significant changes affecting a lot of its essential
features. The introduction of healthcare insurance system,
following the German model, creating professional organizations (Medical College, Pharmacist College etc), the method offinancing services (per capita, DRG, through services), private
initiatives in health, family medicine, reforms of the healthcare
management were key elements for the system.
REFERENCES
1. Joumard I, Andre C, Nicq C. Health Care Systems: Efficiency andInstitutions, Economics Department Working Papers, no. 769, OCED,2010.
2. Smith PC. Market Mechanisms and the Use of Health Care Resources, in Achieving Better Value for money in Health Care, OECD
Health Policy Studies, OECD, Paris, 2008.

SISTEME DE SANATATE IN UNIUNEA EUROPEANA. PREZENTARE DE
ANSAMBLU
REZUMAT
Analiza sistemelor de sanatate existente in statele member ale UE a aratat un mare grad de diversitate, chiar daca toate acestea
sunt inspirate din cele doua sisteme de sanatate model, cunoscute sub denumirile de Beveridge si Bismarck. Sistemele de sanatate
ale UE difera in functie de structura de finantare a sistemului, de gradul de implicare al sectorului de sanatate privat in ansamblul
sistemului de sanatate, de acoperirea pachetului de asigurare, de gradul de libertate in alegerea furnizorilor de servicii, de prezenta
mecanismelor de ghidare spre un anumit tip de ingrijire, etc. Sistemul����������������������������������������������������������������������
de sanatate din Romania a suferit o serie de transformari incepand cu anul 1989, un process care este inca in desfasurare si care este relationat cu lupta pentru democratizare si modernizare
a societatii romanesti.
Cuvinte cheie: sistem de sanatate, UE

2010.20.2(66)  Fiziologia - Physiology

7

CONTEMPORARY MAN IN HIS BIO-PSYCHO-SOCIOECOLOGICAL DIMENSIONSORIN RIGA1, DAN RIGA1, AUREL ARDELEAN2, FRANCISC SCHNEIDER3
Department of Stress Research & Prophylaxis ”Al. Obregia” Clinical Hospital of Psychiatry, Bucharest, RO”V. Goldis” Western University, Arad, RO
3
Center for Applied Physiology and Molecular Biology ”Vasile Goldis” Western University, Arad, RO
1
2

ABSTRACT

Nowadays civilization, globalization and capacity of phenomena understanding make it necessary and imperious redefinition ofhuman notions, concepts and actions. Under the circumstances bio-psycho-social concept of human (Engel - Branzei - Bekhterev)
should be defined in a different manner, namely bio-psycho-socio-ecological dimension of human being (S. Riga - D. Riga - A.
Ardelean - F. Schneider). Consequently bio-psycho-social concept, dimension and determinants of human will be completed withecological ones. This tetrad will have to be present in legislation, human actions and beneficial effects on civilization and environment. Acting in a positive and reasonable manner on geo- and bio-sphere, man will definitely contribute to homeostasis recovery
of Terra system.
Key words: human civilization, redefinition of notions, bio-psycho-social concept of human, new bio-psycho-socio-ecological dimension of human being

INTRODUCTION
In these days, capability of understanding phenomena makes
it imperative and necessary reevaluation of human knowledge,
behaviour and actions. This is why human being should be redefined concerning his concept, significance and determinants
for his future evolution and activity.
Accordingly we surveyed, classified and developed the understanding of human being in its complex structure and build,needs and aspirations. In the course of time, Homo sapiens
received other Latin-based names for a better description ofhuman features and characters. We reviewed the biologic, biosocial, bio-psycho, bio-psycho-social dimensions of humans andintroduced a new concept, namely bio-psycho-socio-ecologicalsignificance of human being.
HUMAN BIOLOGIC DETERMINANT
Human species is defined from Latin as Homo sapiens withsignificance of ”wise man” or ”knowing man”. Scientific classification affiliates the human being to Domain Eukaria; Kindom
Animalia; Phylum Chordata; Class Mammalia; Subclass Theria;
Order Primates; Suborder Haplorrhini; Infraorder Simiiformes;
Parvorder Catarrhini; Superfamily Hominoidea; Family Hominidae; Subfamily Homininae, which contain Genus Homo (humans,
human beings), together with Genera Gorilla (gorillas) and Pan(chimpanzees).
Carl von Linné (Carolus Linnaeus, 1707-1778), Swedish
naturalist, botanist and physician, was the founder of modern
systematics. He elaborated binomial classification system (binomial or binary nomenclature). In naming an organism, Carl von
Linné chose to utilize a two-world naming system (genus nameand specific name or epithet, which together form the species

name), instead of using the full seven-category system (kindomphylum-class-order-family-genus-species). Therefore, human
becomes Homo sapiens as binomial name, in his monumental
monograph Systema Naturae, 10th edition, 1758.
Homo sapiens sapiens (trinomial name introduced by Linnaeus also in 1758 - Genus Homo, Species sapiens, Subspecies
sapiens) is the last representative from his species. He createdhuman history and civilization, and has the possibility towards afascinating evolution and unforeseeable achievements.
HUMAN BIO-SOCIAL DIMENSION
Aristotle-Aristoteles (384 BC - 322 BC) denominated humanbeing as Zóon politikón (Political animal, Homo politicus, Politicalman). Man’s political concept entails man’s social determinant:
The political partnership must be regarded, therefore, as being
for the sake of noble actions, not for the sake of living together(Aristotle, Politics).
During the time humans received other bio-social names:
Homo socius, Social man, man as social being (Peter Berger,
Thomas Luckmann, 1966) and Homo sociologus, Social man(Ralf Dahrendorf).
HUMAN BIO-PSYCHO CHARACTERIZATION
Bio-psycho determinants of human being are very specific,
special and important. They should be divided in features regarding human faculty of reasoning and traits concerning humanemotion and behaviour.
Reason characteristic has multiple significations and denominates Homo sapiens sapiens as:
Animal rationabile, animal capable of rationality (Carlvon Linné 1760, Immanuel Kant 1798);

Received March 2010, Accepted May 2010. Address for correspondence: Dr. Sorin Riga, Department of Stress Research & Prophylaxis, ”Al. Obregia” Clinical Hospital of Psychiatry, 10 Berceni Road,
Sector 4, RO-041914 Bucharest, Romania, e-mail: D_S_Riga@yahoo.com

8

Fiziologia - Physiology  2010.20.2 (66)Homo investigans, Investigating man, human capability
to learn by deduction (Werner Luck 1976);
Homo imitans, Imitating man, human capability to learn
by imitation (A. N. Meltzoff 1988, Jürgen Lethmate 1992);
Homo discens, Learning man, human capability tolearn and adapt (Heinrich Roth, Theodor Wilhelm);
Homo loquens, Talking man, (G. Herder 1772, J. F.
Blumenbach 1779);
Homo faber, Toolmaker man (Benjamin Franklin, KarlMarx, Kenneth P. Oakley, 1949);
Homo creator, Creator man, human creativity (Michel
Landmann 1955, W. E. Mühlmann 1962);
Homo metaphysicum, Metaphysical man (Arthur
Schopenhauer 1819);
Homo religiosus, Religious man (Alister Hardy).
Emotion and behavour describe human being as:- Homo sentimentalis, Sentimental man, man born to a
civilization of sentiment (Milan Kundera
1990, Eugene Halton 1995);- Homo ludens, Playing man, characterization of humanculture as essentially learning thecharacter of play (Friedrich Schiller 1795, Johan Huizinga);- Homo ridens, Laughing man (G. B. Milner 1969);- Homo pictor, Man the artist, human sense of aesthetics(Hans Jonas 1961);- Homo aesthetics, Aesthetic man, capability to appreciateart and beauty (Ellen Dissanayahe
1992);- Homo generosus, Generous man (Tor Nørretranders).
HUMAN BIO-PSYCHO-SOCIAL MODEL
Modern dimension of human associates Homo sapiens sapiens with bio-psycho-social concept. This integrative characterization was elaborated for the first time by V. M. Bekhterev in 1907
(1, 6), then by P. Branzei in 1970 (2, 3) and more recently by G. L.
Engel in 1977 (4, 5). P. Branzei described this human feature asa dynamic and unitary structure respecting psychical processes
in normal and pathological conditions. Right from the start, G.
L. Engel published his new medical - biopsychosocial model ininternational renowned and well-known reviews: Science (1977),
Gen. Hosp. Psychiat. (1979) and Am. J. Psychiat. (1980).
HUMAN BIO-PSYCHO-SOCIO-ECOLOGICALINTEGRATIONWith this paper the authors introduce a new and original
concept about Homo sapiens sapiens, namely humans are andshould be bio-psycho-socio-ecological beings. This integrated
characterization of humans is the result of our critical examination concerning previous human definitions taking into accountthe present stage of human civilization (7, 8). In addition, ourdefinition opens new prospects and possibilities for human future
evolution. Practically, now and in the near future, humans should
be realized a better bio-psycho-social integration associated witha civilized behavior regarding the nature.
2010.20.2(66)  Fiziologia - Physiology

This new complex concept of human being, with his dimensions, features and determinants should be rapidly introducedin legislation (local, Romanian, regional and European), as wellas known and accepted by the population. Then perceptions
and actions of humans regarding the concept will reflect major
modifications in their behaviors and beneficial effects on theenvironment and nature.
In the 20th century, the civilized (?) humans had an uncivilized (!) behaviour respecting the nature, with serious negative
consequences, i.e. lack of legislation for nature protection, severe
pollution, massive forestry clearances, acid rains etc.
In our days and near future (2000-2030) humans must act
as ecological men, in ecological dimensions for an ecologicalcivilization (10, 11). People and countries from the CarpathianDanubian area (Austria, Czech Republic, Poland, Slovakia,
Hungary, Ukraine, Serbia and especially Romania) should havea civilized behaviour regarding the nature.
In this respect, Arad city represents a pattern by ”Vasile
Goldis” Western University (rector prof. dr. Aurel Ardelean). This
university is a model for Euro-regional civilization, an example
which should be followed by others in Romania and Europe.
Here you are three important achievements:
creation and annual organizing of the DKMT (DanubeKris-Mures-Tisa) Euro-regional
Conferences on Environmental Health and Protection;national review Fiziologia-Physiology, Timisoara andArad (editor-in-chief prof. dr. Francisc Schneider), with many
published papers on ecology sciences (9, 13);
present international conference UECDA (Unconventional Energies in the Carpathian and Danubian Area), entitledTechnical and Economical Ecology Issues and the Medical andSocial Aspects (7).
Topics of this UECDA conference are very relevant andimportant because show and indicate solutions and results for:”Green” energy (unconventional and non-pollutant energies, creating energy using biomass, intelligent hydro-energetic
systems);”Green” industry-production-economy (non-pollutant
units, ecological associations and holdings, intelligent hydroenergetic systems);
3R concepts (Reduce, Reuse, Recycle) - waste management hierarchy - EPR (Extended Producer Responsibility).
Contemporary man is an educational project (12) and his
psycho-ecological dimension can be more easily implemented
on human being (14).
CONCLUSIONS
Ecological concept is an important field of globalization, civilization and human evolution. Therefore, humanity must urgently
acts in ecological dimension. The results will be beneficial for
manking and for the Earth too.
Earth Day (April 22) started from 1970 is a good example
to mobilize and unify the ecological forces. Earth Day 2009 isentitled ”Green generation”. In this respect ecological dimension
9

of human being is welcome and should be rapidly introduced in
the behaviour of every person.
REFERENCES
Bio-psycho-social concept
1. Akimenko MA. Vladimir Mikhailovich Bekhterev. J History Neurosci
2007; 16(1-2): 100-109.
2. Branzei P. L’actualité nosologique dans la psychiatrie contemporaine. Critères pour une classification dynamique bio-psycho-sociale
en psychiatrie. Annales Médico-Psycho-logiques (Paris) 1970; 1(4):504-520.
3. Branzei P. Quelques considérations sur la signification d’un concept
tridimensionnel dans le développment de la psychiatrie contemporaine. Annales Médico-Psychologiques (Paris) 1974; 1(3): 341-355.
4. Engel GL. The need for a new medical model: a challenge for biomedicine. Science 1977; 196(4286): 129-136.
5. Engel GL. The biopsychosocial model and the education of healthprofessionals. Gen Hosp Psychiat 1979; 1: 156-165.
6. Neznanov NG, Akimenko MA. Development of a biopsychosocial
concept of disease at the V. M. Bekhterev Institute in the twentiethcentury. Int J Mental Health 2005; 34(4): 4-10.
Bio-psycho-socio-ecological concept
7. Riga S, Riga D, Ardelean A, Schneider F. Contemporary man in

his bio-psycho-socio-ecological dimension. International Conference Unconventional Energies in the Carpathian and Danubian Area ”Technical and Economical Ecology Issues and the Medical and Social Aspects”. Arad, RO, November 27-28, 2009.
8. Riga S, Riga D, Mihailescu A, Motoc D, Mos L, Schneider F. Longevity health sciences and mental health as future medicine. Ann New
York Acad Sci 2009; in press.
Ecology
9. Ardelean A, Craciun C, Farcas T, Soran V. Molecular and cell biology researches as a valuable means to assess the environmental
conditions. Fiziologia-Physiology 2002; 12(4):3-4.
10. Botkin DB, Keller EA. Environmental Science: Earth as a Living
Planet, 7th ed. Ed. Hoboken, NJ, John Wiley & Sons, 2009.
11. Carp R, Gal DG, Muresan S, Preda R. In cautarea binelui comun.
Pentru o viziune crestina a democratiei romanesti. Cluj-Napoca, Ed.
Eikon, 2008.
12. Ilica A. Man is an educational project. Educatia Plus. 2009; 4(1):11-29.
13. Nagymajtenyi L. Environmental effects and the population’s healthstatus. Fiziologia-Physiology. 2002; 12(4): 5-7.
14. Nicola G, Danciu B (eds). Tratat de psihoecologie. Bucuresti, Ed.
Academiei Romane, 2008.

OMUL CONTEMPORAN IN DIMENSIUNEA SA BIO-PSIHO-SOCIOECOLOGICA
REZUMAT

Etapa actuală a civilizatiei umane, globalizarea si capacitatea de intelegere a fenomenelor fac necesara si imperioasa redefinirea
notiunilor, conceptelor si actiunilor umane. In acest context, conceptul bio-psiho-social al omului (Engel - Branzei - Bekhterev)
trebuie definit intr-o alta perspectiva si anume dimensiunea bio-psiho-socio-ecologica a fiintei umane (S. Riga - D. Riga - A. Ardelean- F. Schneider). Astfel, conceptul, dimensiunea si determinantii bio-psiho-sociali ai omului vor fi completati cu cei ecologici. Aceasta
tetrada va trebui sa se regaseasca in legislatie, actiuni umane si efecte benefice asupra civilizatiei si mediului. Actionand pozitiv si
cu discernamant asupra geo- si bio-sferei, omul va contribui decisiv la recapatarea homeostaziei sistemului Terra.
Cuvinte cheie: civilizatie umana, redefinire notiuni, concept bio-psiho-social al omului, noua dimensiune bio-psiho-socio-ecologica
a fiintei umane

10

Fiziologia - Physiology  2010.20.2 (66)REVIEW

INFECTIONS ASSOCIATED WITH PREGNANCY AND
CHILDBIRTHCAMELIA BUDISAN, CONSTANTIN ILIE
Department of Neonatology, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACTPrematurity is the cause of 85% of neonatal morbidity and mortality. Subclinical ascending infections through the lower female genital tract are predominant worldwide. Important micronutrient deficiencies may prevail in low-income countries where these infections
are much more common than in high-income countries. Important morbidities related to poor perinatal outcome both for the motherand for the fetus and newborn comprise preterm birth, prelabor rupture of membranes, placental abruption (predelivery detachmentof the placenta), postpartum sepsis and maternal anemia. In the fetus, sepsis and intrauterine growth retardation are suspected to beconsequences of ascending maternal infections. In the newborn, septicemia and respiratory disorders as well as some neurologicaldisorders seem to be consequences of such ascending genital infections in the pregnant woman. It is concluded that much more
attention should be given to efforts to elucidate the host defense mechanisms and antimicrobial barriers from the vagina throughthe cervix, fetal membranes and amniotic fluid including the early fetal immunocompetence in the second and the third trimester ofpregnancy.
Key words: fetal diseases, maternal morbidity

INTRODUCTION
Infections associated with pregnancy and childbirth have caused
concern for women and their caregivers for centuries. Much attention therefore has been focused on understanding these infections.
Although the clinical approach to infections has improved markedly
in the past few years, infections continue to pose a problem in pregnancy, particularly in low-income countries (1– 4).
Infections are implicated in the pathogenesis of miscarriage,
preterm labor and prelabor rupture of membranes, all of which arecommon events (4). Miscarriage is common worldwide and is theoutcome of approximately 15% of all clinically diagnosed pregnancies. If syphilis and certain vaginal infections are common, thisfigure may reach significantly higher levels, including an increasein miscarriage in the second trimester. Preterm labor may occur in
10–20% of pregnancies in low-income countries whereas prelaborrupture of membranes and postpartum septicemia may occur in
5–10% in such settings. All these in turn are associated with neonatal
infections and morbidity. Both the direct effect of the infection and thematernal immune response contribute to these eventualities (3, 4).
For example, infections that trigger T-helper-1 response can lead tothe release of cytokines such as interferon (IFN)-γ, tumor necrosis
factor (TNF)-α and interleukin (IL)-2 with activation of killer cells andinitiation of preterm labor (3).
Systemic infections and genital infections due to many different
microorganisms including mycoplasmas, Chlamydia trachomatis andTrichomonas vaginalis are reportedly involved in initiating preterm
labor (3,5-9). A wide variety of bacteria present in the normal vaginal
flora of pregnant women such as anaerobes and Escherichia coli canalso cause ascending infections, usually after rupture of membranes,
resulting in intra amniotic infection (10). Chorioamnionitis resulting

from such infections can lead to preterm labor and maternal andfetal morbidity (10). Recent data show that Candida sp. may also
be important in causing preterm labor and neonatal morbidity. intra
amniotic infection due to bacteria in the vaginal flora not only initiatelabor but can also cause infections such as septicemia and meningitis
in the newborn (10, 11).
Several host defense mechanisms operate against ascending infections; these include vaginal acidity, cervical mucus, intact
membranes and antibacterial activity of amniotic fluid (12,13). One
study in India demonstrated that all samples of amniotic fluid inhibited Candida albicans and Clostridium perfringens whereas 50%,
42% and 18%, respectively, inhibited Staphyllococcus aureus, E.
coli and Bacillus fragilis (14). The inhibitory activity could be due
to polymorphonuclear leucocytes, lysozyme, beta lysin, transferrin, immunoglobulins and other bacterial inhibitory factors such as
polypeptide-zinc complexes in amniotic fluid (10).
Intra amniotic infection is difficult to diagnose on the basis ofany single criterion and so diagnosis depends on a set of criteria, themost important clinically being maternal fever and tachycardia andfetal tachycardia(10). The use of laboratory methods for diagnosis
is still not practical. The infection may be polymicrobial, but collecting
amniotic fluid samples without contamination with normal vaginal
flora is cumbersome and may require invasive procedures. Also,
after membrane rupture many bacteria may enter the amniotic cavity
without having caused the rupture. Because of these circumstances,
cultures are not usually attempted, especially in Romania. Recent
literature shows that detection and estimation of surrogate markers
such as C-reactive protein (CRP), cytokines and fetal fibronectin
help in diagnosing intra amniotic infection and in predicting anddiagnosing early-onset neonatal infections (15-18).

Received December 2009. Accepted June 2010. Address for correspondence: Dr. Camelia Budisan, Department of Neonatology, Victor Babes University of Medicine and Pharmacy Timisoara, 2A
Eftimie Murgu Square, e-mail: cbudisan@rdslink.ro2010.20.2(66)  Fiziologia - Physiology

11

Levels of CRP rise when there is a microbial infection or an
inflammation without microbes (19). Studies in pregnant women
showed that CRP is elevated at the onset of labor even in normal
pregnancies and reaches very high levels during the immediate
postpartum period (20). Whether CRP levels are higher than normal
in subclinical infections is not clear and the usefulness of this marker
to diagnose intra amniotic infection remains to be established. However, several studies have shown the usefulness of CRP to predictand diagnose neonatal infections (16-18).
Diagnosis of neonatal septicemia remains a major challenge.
Sepsis can develop in infants with and without risk factors. Clinicalsigns are nonspecific and the laboratory criteria are also not fully
reliable. Although a combination of clinical and laboratory criteria are
required to make a diagnosis, antibiotic treatment is often initiated on
the basis of clinical suspicion alone. Because an infected neonate
can have a negative blood culture, the initiation of antibiotic therapy
without supporting hard evidence of infection is currently justified;in addition, results from blood culture are not available until several
days after the harvesting of blood for culture. Treatment on thebasis of clinical symptoms alone leads to considerable overuse ofantibiotics in the nurseries. Although laboratory data may not be ofmuch use in preventing initiation of therapy, such data could at least
help in stopping unwarranted use of antibiotics.
The tests currently used to diagnose neonatal infections include
total and differential counts, absolute neutrophil count and the ratio of
immature to total white cells. The sensitivity and specificity of thesetests are low. In recent years, CRP estimation has been found to be
useful in diagnosis. One of the pitfalls is that, as mentioned, CRP
can be positive when there is no infection (i.e., the positive predictive
value is very low). To make the predictive values better, a more appropriate cutoff level has to be established. Consensus on the cutoff
level does not exist at present. In true infection, the test may become
positive after 12 h, so estimation of CRP at presentation may not be ofmuch value in diagnosis. Serial determinations may be required andmay have a better predictive value than static single estimates (21).
This test may be valuable for making decisions about discontinuingtherapy. The test can be done using automated systems and a latex
agglutination test, which is widely available in Romania.
Over the years several proinflammatory cytokines have beentested for their use in diagnosing IAI and neonatal infections.
These cytokines include IL-2, IL-6, IL-8 and IFN-γ. Maternal, cord
and neonatal blood IL-6 levels have been found to correlate withchorioamnionitis and neonatal sepsis (16-18).
IL-6 stimulates the production of CRP. Therefore, IL-6 levels
should rise before CRP levels rise. Several studies have confirmedthat IL-6 is an early and sensitive marker of sepsis in newborns andin adults. IL-6 levels are found to be better predictors of mild sepsis(22). Combined use of IL-6 and CRP is found to give better predictive
values than the use of either alone. However, more studies in different
settings are required to confirm these findings and to evaluate theirapplicability as routine diagnostic tests.
TNF-α is responsible for organ injury. Although the levels of thiscytokine also increase in infection, this is a less sensitive marker
than IL-6. Combined use again increases sensitivity (22). IL-1β is asoluble protein released by macrophages in response to infection
12and inflammation. With IL-6 and TNF-α it also can initiate acutephase responses such as fever and synthesis of acute phase hepatic
proteins such as CRP. However, estimation of levels of this cytokine
in infections has yielded conflicting results and it is not considered
important for diagnosis (22). Another widely studied marker is fetal
fibronectin. Elevated levels of fetal fibronectin in vaginal fluids ishighly predictive for preterm labor. This marker is detected with theuse of monoclonal antibodies (19, 20).
Prelabor rupture of membranes.
The term “prelabor” should be used rather than “premature” or“preterm” because the latter two relate neither to gestational age nor
to the weight of the fetus or neonate. The membrane rupture itself
should be characterized as preterm (occurring before 259 completeddays) or term (occurring after 259 completed days).
Several studies have shown that in patients with prelabor ruptureof membranes in the preterm period, prophylactic antibiotics are ofvalue in prolonging the latent period between rupture and onsetof labor and in reducing the incidence of maternal and neonatal
infection (32). The most extensively tested antibiotic regimen used
for prophylaxis includes erythromycin either alone or with ampicillin(32). There is no evidence that antibiotic therapy prevents prelaborrupture of membranes. Bacterial vaginosis in early pregnancy has
been found to be associated with prelabor rupture of membranes
in the preterm period (31).
Considerable attention has been given to ILs as predictors ofprelabor rupture of membranes. Lewis et al. (37) found that IL- 6 inmaternal plasma was a predictor of neonatal infectious complications
in patients with prelabor rupture of membranes even when the data
were stratified for patients receiving and not receiving corticosteroids.
The neonatal infectious complications examined included respiratory distress syndrome, necrotizing enterocolitis, intraventricularhemorrhage, IAI, presumed neonatal sepsis, neonatal sepsis andcongenital pneumonia.
Reactive oxygen species, which are generated by the body’s
response to diverse insults such as infection, have also attracted attention. Such insults may activate collagenolytic enzymes and impair
fetal membrane integrity (38). This impairment is then inhibited byantioxidants like vitamin E and possibly vitamin C (38). Damage byreactive oxygen species that impairs fetal membrane integrity andreduces midgestation levels of vitamin C is associated with prelaborrupture of membranes in the preterm period (38). Vitamins E and C
can be safely and effectively absorbed and delivered to gestational
tissues, which opens the possibility of intervention trials (38).
Fetal morbidity
Fetal sepsis - studies on cord blood in women with clinicalsuspicion of having infants subject to IAI have shown that cord
blood cytokines may predict neonatal outcome. Cord blood fromneonates with intrauterine infections had more IFN-γ. – producing
CD3+T cells than did cord blood from uninfected neonates (45). The
percentage of these cells in the infected neonates correlated with aduration of membrane rupture before the onset of labor but not withthe level of CRP. The infected neonate born the longest time after
membrane rupture had an increased percentage of IL-4–producing
Fiziologia - Physiology  2010.20.2 (66)CD3+T cells. The result suggests that the increase of cord blood
IFN-γ – and IL-4 – producing T cells is part of the immune system’s
reaction to perinatal intrauterine infections (45).
Intrauterine growth retardation - most of the literature available
linking infection with intrauterine growth retardation focuses onmalaria. Some evidence shows that cytomegalovirus infections may
play a role in this context. Cytomegalovirus immunoglobulins were
given to pregnant women with primary cytomegalovirus infection to
inhibit viral activity; the authors concluded that this treatment may
prevent fetal cytomegalovirus infection (46). A study from India did
not substantiate any relationship between cytomegalovirus infection
and intrauterine growth retardation (47).
Neonatal morbidity
Neonatal sepsis. As discussed above, neonatal blood IL-6
levels have been found to correlate with chorioamnionitis andneonatal sepsis (16-18).
Neonatal respiratory disorders. Several studies now correlate
intrauterine infection and neonatal respiratory disorder. The link between chorioamnionitis and intrauterine lung injury with subsequentdevelopment of bronchopulmonary dysplasia has been substantiated (48). Exposure to pro-inflammatory cytokines is implicated inthis impairment of the fetal lung. Hitti et al. (49) demonstrated thatin amniotic fluid infection, elevated TNF - is associated with respiratory distress syndrome, multiple organ dysfunction and various
intracerebral disturbances.
Neonatal neurological disorders squeals (49), such as intraventricular hemorrhage and multiple organ dysfunctions. Similar
results were shown in other studies and evidence now exists of a
relationship between intrauterine infection and the development of
neonatal intraventricular hemorrhage, possibly by the ventricularleukomalacia with subsequent cerebral palsy (48, 50). The intraventricular hemorrhage is thought to be mediated through the generationof pro-inflammatory cytokines by the fetus.
CONCLUSION
Only partially understood host defense mechanisms operate
against infections affecting maternal and fetal morbidity. Subclinicalascending infections through the lower female genital tract are predominant worldwide. Proinflammatory cytokines have been tested
for their use in diagnosing such infections, and promising leads
indicate that affordable kits may soon be available for serologicaldiagnosis of the mother. Important morbidities related to poor
perinatal outcome both for the mother and the fetus and newborn
comprise preterm birth, prelabor rupture of membranes, placental
abruption, postpartum sepsis and maternal anemia. Fetal sepsis andintrauterine growth retardation are suspected to be consequencesof ascending maternal infections. Neonatal septicemia and neonatal
respiratory disorders as well as some neurological disorders seem tobe consequences in the newborn of such ascending genital infectionsin the pregnant woman.
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JD, Coultrip L, Eriksen N, Holbrook H, Elliot J, Ingardia C, Pietrantoni M.
Fetalfibronectin as a predictor of preterm birth in patients with symptoms:
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20. Crane JMG, Armson A, Dodds L, Feinberg RF, Kennedy W, Kirkland
SA. Risk scoring, fetal fibronectin and bacterial vaginosis to predict preterm delivery. Obstet. Gynecol. 1999; 93: 517-522.
21. Kawamura M, Nishida H. The usefulness of serial C-reactive protein
measurement in managing neonatal infection. Acta Paediatr. 1995; 84:13

10-13.
22. Vesikari T. Cytokine determinations and rapid diagnosis of early onset
neonatal septicaemia. Acta Pediatr. 1999; 88: 585-591.
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Hederstedt B. Prevalence of syphilis infection in Mozambican women withsecond trimester miscarriage and women attending antenatal care in second trimester. Genitourin. Med. 1993; 69: 431-433.
24. Samucidine M, Barreto J, Folgosa E, Mondlane C, Bergström S. Infertile women in developing countries: at potentially high risk of HIV transmission. Afr. J. Reprod. Health 1999; 3: 98-102.
25. Mathai E, Mathai M, Prakash JA, Bergström S. Audit of managementof pregnant women with positive VDRL test. Natl. Med. J. India 2001; 14:
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26. Lockwood CJ, Kuczynski E. Markers of risk for preterm delivery. J.
Perinat. Med. 1999; 27: 5-20.
27. Challis JR, Lye SJ, Gibb W, Whittle W, Patel F, Alfaidy N. Understanding preterm labor. Ann. N. Y. Acad. Sci. 2001; 943: 225-234.
28. McLean M, Bisits A, Davies J, Woods R, Lowry P, Smith R. A placental
clock controlling the length of human pregnancy. Nat. Med. 1995; 1: 460463.
29. Van Meir CA, Sangha RK, Walton JC, Mathews SG, Keirse MJ, Challis
JR. Immunoreactive 15–hydroxyprostaglandin dehydrogenase (PGDH) is
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30. Lei H, Furth EE, Kalluri R, Chiou T, Tilly KI, Tilly JL, Elkon KB, Jeffrey
JJ, Strauss JF. A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor. J. Clin. Invest.
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vaginosis in early pregnancy and adverse pregnancy outcome. J. Obstet.
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58-64.
34. McDonald HM, Chambers HM. Intrauterine infection and spontaneous
midgestation abortion: is the spectrum of microorganisms similar to that inpreterm labor? Infect. Dis. Obstet. Gynecol. 2000; 8: 220-227.
35. Lu GC, Schwebke JR, Duffy LB, Cassell GH, Hauth JC, Andrews WW,
Goldenberg RL. Midtrimester vaginal Mycoplasma genitalium in womenwith subsequent spontaneous preterm birth. Am. J. Obstet. Gynecol.
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program for preventing prematurity. J. Perinat. Med. 2001; 29: 199-211.
37. Lewis DF, Barrilleaux PS, Wang Y, Adair CD, Baier J, Kruger T. Detection of interleukin–6 in maternal plasma predicts neonatal and infectious
complications in preterm premature rupture of membranes. Am. J. Perinatol. 2001; 18: 387-391.
38. Woods JR, Plessinger MA, Miller RK. Vitamins C and E: missing links
in preventing preterm premature rupture of membranes? Am. J. Obstet.
Gynecol. 2001; 185: 5-10.
39. Lockwood CJ, Krikun G, Aigner S, Schatz F. Effects of thrombin onsteroid-modulated cultured endometrial stromal cell fibrinolytic potential. J.
Clin. Endocrinol. Metab. 1996; 81: 107-112.
40. Elovitz MA, Saunders T, Ascher-Landsberg J, Phillippe M. Effects ofthrombin on myometrial contractions in vitro and in vivo. Am. J. Obstet.
Gynecol. 2000; 183: 799-804.
41. Libombo A, Folgosa E, Bergström S.Risk factors in puerperal endometritis-myometritis. An incident case-referent study. Gynecol. Obstet.
Invest. 1994; 38: 198-205.
42. Bergström S, Libombo A. Low birthweight and post partum endometritis-myometritis. A case-referent study. Acta Obstet. Gynecol. Scand. 1995;
4: 611-613.
43. Libombo A, Folgosa E, Bergström S. A case-referent study on postcaesarean endometritis-myometritis in Mozambique. Gynecol. Obstet.
Invest. 1995; 39: 180-185.
44. Van den Broek NR, Letsky EA. Etiology of anemia in pregnancy insouth Malawi. Am. J. Clin. Nutr. 2000; 72: 247S-256S.
45. Matsuoka T, Matsubara T, Katayama K, Takeda K, Koga M, Furukawa
S. Increase of cord blood cytokine-producing T cells in intrauterine infection. Pediatr. Int. 2001; 43: 453-457.
46. Cosmi E, Mazzocco M, La Torre R, Ligi P, Sali E, Nigro G. Therapy
or prevention of fetal infection by cytomegalovirus with immunoglobulin
infusion in pregnant women with primary infection. Acta Biomed. Ateneo
Parmense 71 (Suppl. 1), 2000; 547-551.
47. Deorari AK, Broor S, Maitreyi RS, Agarwal D, Kumar H, Paul VK, Singh
M. Incidence, clinical spectrum, and outcome of intrauterine infections in
neonates. J. Trop. Pediatr. 2000; 46: 155-159.
48. Vigneswaran R. Infection and preterm birth: evidence of a common
causal relationship with bronchopulmonary dysplasia and cerebral palsy.
J. Paediatr. Child Health 2000; 36: 293-296.
49. Hitti J, Tarczy-Hornoch P, Murphy J, Hillier SL, Aura J, Eschenbach DA.
Amniotic fluid infection, cytokines, and adverse outcome among infants at
34 weeks’ gestation or less. Obstet. Gynecol. 2001; 98: 1080-1088.
50. Svigos JM. The fetal inflammatory response syndrome and cerebral
palsy: yet another challenge and dilemma for the obstetrician. Aust. N. Z.
J. Obstet. Gynaecol. 2001; 41: 170-176.

INFECTII ASOCIATE CU SARCINA SI NASTEREAREZUMAT
85% dintre cauzele de morbiditate si mortalitate sunt asociate cu prematuritatea. In intreaga lume sunt predominante infectiile
subclinice ascendente de la nivelul tractului genital feminin. In tarile subdezvoltate pot sa predomine deficitele nutritive importante,aceste tipuri de infectii fiind mult mai raspandite comparativ cu tarile dezvoltate. Intre morbiditatile importante relationate cu evolutie
perinatala deficitara, atat a mamei cat si a fetusului si non-nascutului, se afla ruptura de membrane inainte de travaliu, nastere
inainte de termen, detasarea placentei, sepsis post-partum si anemia materna. La fat, sepsisul si retardul in dezvoltarea intrauterinasunt suspectate ca fiind consecinta infectiilor ascendente materne. La nou-nascuti, septicemia si afectiunile respiratorii, precum si
dereglarile neurologice par sa fie tot consecinta unor astfel de infectii genitale materne, in perioada sarcinii. Se poate concluziona
ca trebuie acordata atentie sporita eforturilor de elucidare a mecanismului de aparare si barierelor antimicrobiene de la nivelul
vaginului pana la cervix, membranelor fetale si fluidului amniotic, incluzand imunocompetenta fetala precoce, in trimestrele doi si
trei de sarcina.
Key words: afectiuni fetale, morbiditate materna

14

Fiziologia - Physiology  2010.20.2 (66)

C REACTIVE PROTEIN IN DIABETES MELLITUS TYPE 2
COMPLICATED WITH DIABETIC NEPHROPATHY
MATEI D1, MOLDOVAN C1, MURESAN M1, MICLE L1, MICLE O1, BURTA O1, OROS R1,
MURESAN A2
1
Faculty of Medicine and Pharmacy Oradea
2
Physiology Department, University of Medicine and Pharmacy I. Hatieganu, Cluj- Napoca

ABSTRACT
Aims: In subjects with diabetes mellitus type 2 complicated with diabetic nephropathy, there is an increase of markers of inflammation, generated by multiple mechanisms. Chronical subclinical inflammation is a finding observed in diabetes mellitus type 2. This
study was designed to establish the correlation between the level of inflammatory marker C-reactive protein (CRP) with the durationof diabetes, the degree of glycemic control and the stage of diabetic nephropathy. Method: The study was performed on four groupsof subjects suffering from diabetes mellitus type 2 complicated with nephropathy and a control group. The first group included 47
patients with diabetes mellitus type 2 and normoalbuminuria (UAE < 30mg/d), the second group included 54 subjects with diabetes
and microalbuminuria (UAE = 30 – 300mg/d), the third group had 49 subjects with diabetes type 2 and macroalbuminuria (UAE>
300mg/d). In the forth group were selected 45 patients with macroalbuminuria and chronic renal failure. In the control group were
included 25 subjects. For all individuals included in the study we assessed the following parameters: glycemia, glycosylated hemoglobin A1c, creatinine, urea, uric acid, glomerular filtration rate, C reactive protein and urinary albumin excretion/day. Results: We
obtained a significant increase of these elements in study groups by comparison with control group, except the glomerular filtration
rate, which decreased. CRP was significant increased in experimental groups comparatively with the control group (p<0.001). We
found a positive significant correlation between the level of CRP and UAE (r = 0.49, p< 0.001). The correlation of HbA1c with CRP
was r = 0.25, p < 0.001 and the correlation between the concentration of CRP and the duration of diabetes r = 0.31, p<0.001, both
positive and significant statisticaly. Conclusions: The level of CRP was significantly elevated in diabetic patients complicated withnephropathy and it correlates positively with the urinary excretion of albumin, the duration of diabetes mellitus and the degree ofglycemic control. In addition to metabolic and hemodynamics factors, inflammation can play a role in pathogenesis of diabetes mellitus type 2, complicated with nephropathy.
Keywords: diabetes mellitus type 2, diabetic nephropathy, C reactive protein, urinary excretion of albumin.

INTRODUCTION
In the recent years numerous studies have shown that lowgrade inflammation is associated with the risk of developing
type 2 diabetes mellitus and its vascular complications (1, 14).
Patients with diabetes type 2 and nephropathy exhibit high levelsof different acute phase markers of inflammation: C-reactive
protein (CRP), fibrinogen, serum amyloid A and IL-6. High levelof glycemia contributes to the glycation of proteins and lipids,
resulting in formation of advanced glycated end products (AGE).
The receptors for AGE are expressed in different tissues andcells, including endothelial cells, smooth muscle cells and macrophages. The binding of AGE to their specific receptors leads
to intracellular generation of reactive oxygen species (ROS),
which in turn activates the nuclear factor-kappa B ( Nf-kB). A
consequence of this fact is the increase of expression of multiple
cytokines (Tumor necrosis factor alpha and beta, Interleukins
1, 6, 8, 18 and gamma interferon). Especially IL 6 and TNFα
stimulate the synthesis of CRP, complement, serum amyloidA, fibrinogen, von Willebrand factor, cortisol and plasminogenactivator inhibitor-1( 9, 17, 18 ). Subjects with kidney failure
or uremia manifest evidence of chronic inflammation (17, 22).

Chronic inflammatory process is thought to play an importantrole in development of micro and macrovascular complications intype 2 diabetes mellitus (1). The inflammatory marker C-reactive
protein (CRP) has been found in most prospective studies to be
associated with future cardiovascular outcomes (11).
The aim of this study was to establish the correlation existing
between the level of inflammatory biomarker C- reactive protein
and the duration of diabetes type 2, the degree of glycemic
control and the stage of diabetic nephropathy. We hypothesizedthat in patients with diabetes mellitus type 2, complicated withnephropathy, the urinary excretion of albumin might be related
to the marker of chronic inflammation (CRP) and there is a positive correlation between the duration of diabetes, the quality ofglycemic control and the concentration of CRP.
MATERIAL AND METHOD
We performed the study on four groups of patients sufferingof diabetes mellitus type 2 with diabetic nephropathy admitted
at The Municipal Hospital from Oradea and one control groupof healthy subjects.
The first study group included 47 patients with diabetesReceived March 2010. Accepted May 2010. Address for correspondence: Matei Daniela, University of Oradea, Faculty of Medicine and Pharmacy, Department Preclinic I, no.10 P-ta 1 Decembrie St.,
410037, Oradea, Romania, phone + 40(259)415680, Fax. +40(259)418266, e-mail: danielacernau@yahoo.com

2010.20.2(66)  Fiziologia - Physiology

15

RESULTS
We obtained a significant increase of all assessed parameters in study groups by comparison with control group.
The subjects of experimental groups had higher values of glycemia, glycosilated hemoglobin A1c, uric acid, serum creatinine,urea and C reactive protein, except the glomerular filtration rate
16

which was lower in these groups than in control group.
The values of glycosylated hemoglobin A1c was 7.32 ± 1.32
% in group 1 (normoalbuminuria), 7.84 ± 1.36% in the second
group (microalbuminuria), 8.33 ± 1.69 % in the third group (macroalbuminuria) and 8.05 ± 1.89 % in the last experimental group(macroalbuminuria and chronic renal failure). The mean value ofglycosylated hemoglobin in control group was 5.23 ± 0.64%.
C Reactive Protein presented a similar variation, with higher
values in experimental groups compared with control group. It was
4.40 ± 1.19 mg/dL in group 1, 6.48 ± 2.75 mg/dL in group 2, 7.36 ±3.50 mg/dL in group 3 and 13.40 ± 13.00 in the group 4. The control
group had a mean value of 2.98 ± 0.61 mg/dL. This inflammatory
marker had a statistically significant increase related to the control
group (p < 0.001) for all experimental groups (Figure 1).
Values of CRP in the study and control groups

13,4

14
12
10
mg/L

8

6,48

6

7,36

4,4

4

2,98

2
0
CRP

CRP gr. 1 CRP gr. 2 CRP gr. 3 CRP gr. 4

c ontrol gr.

Fig. 1. Values of CRP in the study and control groups

The correlation between the duration of diabetes mellitus andthe concentration of CRP was r = 0.31, p< 0.001 (Figure 2). The
concentration of glycosylated hemoglobin A1c had a positive significant correlation with CRP, r = 0.25, p = 0.001 (Figure 3). Between
the level of CRP and urinary albumin excretion there was also apositive significant correlation (r = 0.49, p < 0.001) (Figure 4).
50
45
40
35
C reacive protein

mellitus type 2 and normoalbuminuria (UAE< 30 mg/d). The
characteristics of this group were: the mean age – 53.53 ±8.77 years, the distribution on sexes- 34.04% female and65.96% male, mean duration of diabetes 4.83 ± 2.66 years,
BMI (body mass index) = 29.54 ± 4.62 kg/m2, 55.31% of themwere insulin-dependent and 44.69 % non insulin-dependent(treated with biguanides and sulhonylureas). In the second
group were admitted 54 patients with diabetes mellitus type
2 and microalbuminuria (UAE = 30 – 300 mg/d). In this group44.44 % of patients were female and 55.56% male, the mean
age was 61.37± 7.67 years, the BMI = 30.96 ± 4.72 kg/m2, themean duration of diabetes 8.07 ± 3.17 years, 62.96 % insulin
dependent and 37.04 % non insulin-dependent. The third group
included 49 subjects with diabetes mellitus and macroalbuminuria (UAE> 300 mg/d), having the following characteristics:
the mean age: 64.86 ± 7.69 years, mean duration of diabetes
10.24 ± 4.45 years, BMI = 29.86 ± 3.96 kg/m2, 42.86% female
and 57.14% male, 95.91% insulin dependent. In the last groupwere included 45 subjects, with macroalbuminuria and chronic
renal failure (GFR < 60 mL/min/1.73m2), having the age of 65.22± 6.74 years and a mean duration of diabetes of 13.84 ± 3.87
years, 42.22% were female and 57.78% male. Their BMI was
29.40 ± 5.47 kg/m2, all insulin dependent.
In the control group we selected 25 subjects matched forage, sex and BMI with the patients of study groups (mean age= 49.32 ± 3.97 years, 40% female and 60 % male and BMI=
28.16 ± 4.20 Kg/m2).
Blood samples were collected in the morning, after 8-12 hoursof overnight fasting. We analyzed glycemia, glycosylated hemoglobin A1c, glomerular filtration rate, serum creatinine, urea, uric acid, C
reactive protein (CRP), complete blood count, urine analysis. Two
24 hours samples of urine were collected from the subjects, from
where UAE was confirmed and the mean value was calculated.
Glomerular filtration rate was calculated with MDRD formula withfour variables: creatinaemia, age, sex, and race.
Urinary albumin excretion was assessed by Micro-Albumin
ELISA test (DRG Diagnostics), glycosilated hemoglobin by achromatographic- colorimetric method (Biogamma) and CRP
by Turbilatex method (Al Instruments KFT).
According to our inclusion criteria, patients with urinary tract
infection or other infections, inflammatory disease, pyrexia,
malignancy, active immunological diseases, were excluded fromthe study. All of the subjects participating in this study provided
the written informed consent.
The statistical analysis of data was performed by SPSS 13.0.
The correlation between the mentioned parameters was tested
by Pearson`s correlation test. Differences in the parameters
between the groups were analyzed by Student`s test.

30
25
y = 0,4158x + 3,9977
R2 = 0,0701

20
15
10
5
0
0

5

10

15

20

25

30

Duration of Diabetes

Fig. 2. Correlation of the duration of diabetes with CRP

Fiziologia - Physiology  2010.20.2 (66)

50
45

C Reactive Protein

40
35
30
25
y = 0,9378x + 0,122
R2 = 0,0533

20
15
10
5
0
3

4

5

6

7

8

9

10

11

12

13

Glycosylated Hemoglobin

Fig. 3. Correlation of Glycosylated hemoglobin A1c with CRP in diabetic
subjects
50
45

C Reactive Protein

40
35
30
25
20
y = 0,003x + 5,2683
R2 = 0,2169

15
10
5
0
0

1000

2000

3000

4000

5000

6000

7000

Urinary Albumin Excretion/day

Fig. 4. Correlation of CRP with UAE in diabetic subjects

DISCUSSION
Diabetic nephropathy is considered a progressive glomerular disease, having a predominant inflammatory nature,
which evolves in diabetic medium. It has been suggested that
long- term innate immune system activation, resulting in chronic
inflammation, elicits disease instead of having a benefic effect,
which can lead to development of insulin resistance syndrome
and diabetes mellitus type 2. Pro-inflammatory cytokines andacute phase reactants are involved in multiple metabolic pathways relevant to insulin resistance, including insulin regulation,
reactive oxygen species, lipoprotein lipase action and adipocyte
function (7).
Certain clinical and experimental studies have demonstrated
that in diabetic nephropathy there are signs of inflammation,
which play a significant role in pathogenesis of this disease.
Chow et al. (6) showed that db/db mice, a model of type 2 diabetes and diabetic nephropathy, exhibited an increased expressionof intracellular adhesion molecule-1 (ICAM-1), which stimulates
2010.20.2(66)  Fiziologia - Physiology

inflammation by increasing leukocyte infiltration and adherence inglomeruli and tubules, along with macrophage infiltration. Hasegawa et al. (12) demonstrated that macrophages incubated withglomerular basement membranes from diabetic rats produced
greater levels of IL-1 and TNF-α, than macrophages incubatedwith membranes of normal rats. IL-1 increases vascular endothelial permeability and has been involved in the proliferation ofmesangial cells and matrix synthesis, as well as in the development of intraglomerular microcirculatory abnormalities.
Markers of inflammation, a well- recognized manifestationof oxidative stress have been observed to increase in responseto elevated glucose levels (26). It has been proposed that type2 diabetes mellitus is a disease of immune system, involving acytokine-mediated acute phase inflammatory response (20).
In this disease there is an accelerated rate of atherosclerosis,
which is though to be due in part to the irreversible formationand deposition of molecules known as advanced glycation end
products (AGEs). Augmented blood levels of glucose contributeto the glycation of proteins and lipids, resulting in the formationof AGEs (2). Reaction of AGEs with the receptors for AGEs
(RAGEs) generates reactive oxygen species (ROS), responsible
for the activation of signal transduction cascade (PKC, MAPK
and JAK/STAT) and transcription factors (NF-kB, AP-1) and
upregulate TGF-β1 and fibronectin in renal cells. ROS-regulated
signaling pathways lead to extracellular matrix (ECM) depositionin diabetic kidney. In addition to upregulation of ECM synthesis,
ROS play an important role in ECM degradation and epithelialmesenchymal transition in tubular epithelial cells leading toglomerular mesangial and tubulo-interstitial expansion (16).
Increase of acute –phase markers are associated withincrease cardiovascular risk, because chronic inflammationis one of the pathogenetic mechanisms of atherosclerosis. In
contrast, the relationship between low-grade inflammation anddiabetic microangiopathy is still unclear. As regard the diabetic
nephropathy several studies have examined the relationships
with inflammation, leading to conflicting results (20, 22, 25). Most
studies have reported an increase in acute-pahse biomarkers inpatients with diabetes and microalbuminuria. The coexistenceof an inflammatory condition with diabetic nephropathy could
explain in part the tremendously increased cardiovascular risk
among these patients (15, 21, 24). Dalla Vestra et al. (8) showedthat patients with diabetic nephropathy exhibit high level ofdiverse acute-phase markers of inflammation, such as CRP,
fibrinogen, serum amyloid, interleukin 6 and these are correlatedwith the thickening of glomerular basement membrane.
In our study we found that there was an increase of level ofinflammatory marker – CRP in patients with diabetes mellitus
type 2 than controls (p< 0,001); furthermore the values of thisparameter increased significantly as nephropathy progressed.
CRP had a positive significant correlation with the urinary excretion of albumin (r= 0.49, p < 0.001). Similar results were obtainedby other researchers. Navarro et al. found a correlation between
CRP and UEA of r = 0.68, p < 0.001. They also established apositive correlation of CRP with duration of diabetes and glycatedhemoglobin level (19). Streja et al. indicated the same positive17

correlation between these parameters (25). The prospective
Irbesartan Diabetic Nephropathy study concluded that CRP was
positively associated with female sex, BMI, serum creatinine, hemoglobin A1c and inversely correlated with pyridoxal 5`-phosphateand folate. But it revealed that in patients with nephropathy,
CRP does not add predictive information above and beyond
that provided by traditional established risk factors. A finding ofa link between CRP and congestive heart failure needs further
confirmation (5).The Insulin Resistance Atherosclerosis Study
demonstrated a significant association between inflammatory
markers (CRP and fibrinogen) and urinary albumin excretion in
type 2 diabetic patients with microalbuminuria (10). Choudhary
et al. (2008) found a positive correlations between the level of
CRP and urinary albumin excretion (r = 0.781, p < 0.001) and
CRP and glycosylated hemoglobin ( r = 0.750, p < 0.001) .
In patients with diabetes mellitus the incidence of macroangiopathy is at least twice higher than in nondiabetic subjects.
Although the concept of atherosclerosis as an inflammatory
disease is now well established, line of evidence suggests thatchronic inflammation may be involved in the pathogenesis ofinsulin resistance and diabetes mellitus type 2. This leads tothe hypothesis that inflammatory changes may be considered
as common pathogenetic step in all these conditions. Oxidative
stress may explain the presence of inflammation in all these
conditions. The pathways that generate the mediators of inflammation, such as adhesion molecules and interleukins are all
induced by oxidative stress. (4) Elevated concentration of inflammatory markers may be the result of preexisting atherosclerosis
in patients with abnormal albuminuria. On the other hand, theinflammatory markers may alter the glomerular function andthus be causally involved in the development of albuminuria. In
diabetic nephropathy there is an infiltration of glomeruli and interstitium with macrophages, which can responsible for increased
level of inflammatory biomarkers (5).
Higher level of inflammatory parameter (CRP) in diabetes
type 2 complicated with nephropathy indicates that inflammation
might be a pathogenetic mechanism in this condition, but further
studies are necessary to confirm the intra-renal production of
inflammatory cytokines. Treatment with anti-inflammatory drugs
might be beneficial in preventing the progression of diabetic
nephropathy from its initial stage towards the renal failure, due
to the new vision of pathogenesis of diabetes mellitus.
CONCLUSIONS
1. The concentrations of CRP was increased in patients withdiabetes mellitus type 2 complicated with nephropathy and its
value was higher as long as diabetes progressed
2. There was a positive significant correlation between thelevel of CRP and urinary excretion of albumin
3. There was a positive correlation between the concentrationof CRP and duration of diabetes mellitus
4. CRP showed a positive correlation with the level of glycemic control (glycosylated hemoglobin A1c )5. Inflammation could be a pathogenetic mechanism responsible for alteration of glomerular function, involved in the18

development of albuminuria
It is possible that in future new therapeutic strategies basedon anti-inflammatory drugs to be effective in treatment of diabetic
complications
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18. Moriwaki Y, Yamamoto T, Shibutani Y, et al. Elevated levels of interleukin 18 and tumoral necrosis factor- alpha in serum of patientswith diabetes mellitus: relationship with diabetic nephropathy. Metabolism. 2003; 52:605-8.
19. Navarro JF, Mora C, Maca M, Garca J. Inflammatory parameters
are independently associated with urinary albumin in type 2 diabetes
mellitus. Am J Kidney Dis. 2003; 42(1): 53- 61.
20. Pickup CJ. Inflammation and activated innate immunity in thepathogenesis of type 2. Diabetes Care 2004; 27:813-23.
21. Sahareimo M, Teppo A, Forsblom C, Fagerudd J, Groop PH. Diabetic nephropathy is associated with-grade inflammation in type 1 diabetic patients. Diabetologia 2003; 46:1402-7.
22. Schalwijk CG, Poland D, van DijkW, et al. Plasma concentrationof C-reactive protein is increased in type 1 diabetic patients withoutclinical macroangiopathy and correlates with markers of endothelial

dysfunction:Evidence for chronic inflammation. Diabetologia 1999;
42:351-357.
23. Shlipak MG, Fried LF, Crump C, et al. Elevations of inflammatory
and procoagulant biomarkers in elderly persons with renal insuffiency.
Circulation 2003; 170:87-92.
24. Stehouwer CD, Gall M, Twisk J, Knudsen E, Emeis J, Parving
H. Increased urinary albumin excretion, endothelial dysfunction andchronic low-degree inflammation in type 2 diabetes: Progressive, interrelated and independently associated with risk of death. Diabetes
2002; 51: 1157-65.
25. Streja D, Cressey P, Rabkin SW. Associations between inflammatory markers, traditional risk factors and complications in patients with
type 2 diabetes mellitus. J Diabetes Complications 2003; 17:120-127.
26. Wright E, Scism-Bacon JL, Glass LC. Oxidative stress in type 2
diabetes: the role of fasting postprandial glycemia. Int J Clin Pract
2006; 60(3):308-314.

PROTEINA C REACTIVĂ ÎN DIABETUL ZAHARAT TIP 2 COMPLICAT CU
NEFROPATIE DIABETICĂREZUMAT
Obiective: La subiecţii cu diabet zaharat tip 2, complicat cu nefropatie dibaetică există o creştere a markerilor inflamatori, generaţi
prin mecanisme multiple. Inflamaţia cronică subclincă a fost observată în diabetul zaharat de tip 2. Scopul studiului a fost de a stabili
corelaţia dintre nivelul markerului inflamator - Proteina C reactivă (PCR) cu durata diabetului, gradul controlului glicemic şi stadiul
nefropatiei diabetice.
Material şi Metodă: Studiul a fost efectuat pe patru grupe de subiecţi cu diabet zaharat tip 2 complicat cu nefropatie şi un grup decontrol. Primul grup experimenatal a cuprins 47 de pacienţi cu diabet zaharat tip 2 şi normoalbuminurie ( UAE < 30mg/zi), al doilea
grup a inclus 54 de subiecţi cu diabet zaharat şi microalbuminurie ( UAE = 30- 300mg/zi), cel de al treilea grup 49 de subiecţi cu
diabet şi macroalbuminurie (UAE >300 mg/zi). În al patrulea grup au fost selectaţi 45 de pacienţi cu macroalbuminurie şi insuficienţă
renală cronică. În grupul martor au fost selectaţi 25 de subiecţi. Pentru toţi subiecţii incluşi în studiu am determinat următorii paramentri: glicemia, hemoglobina glicozilată A1c , creatinina, urea, acidul uric, rata de filtrare glomerulară, proteina C reactivă şi excreţia
urinară de albumină/24 ore. Rezultate: Am obţinut o creştere semnificativă a acestor paramentri în grupele de studiu în comparaţie
cu lotul martor, cu excepţia ratei de filtrare glomerulară, care a scăzut. PCR a fost semnificativ crescută la loturile experimentalefaţă de lotul martor (p <0,001). Am stabililit o corelaţie pozitivă şi semnificativă între concentraţia PCR şi UAE (r = 0,49, p< 0,001).
Corelaţia HbA1c cu PCR a fost de r = 0,25, p < 0,001, iar corelaţia dintre nivelul PCR şi durata de evoluţie a diabetului zaharat r =0,31, p< 0,001, ambele pozitive şi semnificative statistic. Concluzii: Nivelul PCR a fost semnificativ crescut la subiecţii cu diabet
zaharat tip 2 complicat cu nefropatie şi s-a corelat pozitiv cu excreţia urinară de albumină, durata şi evoluţia diabetului zaharat, cât şi
cu gradul controlului glicemic. Pe lângă factorii metabolici şi hemodinamici, inflamaţia ar putea deţine un rol în patogeneza diabetului
zaharat tip 2 complicat cu nefropatie.
Cuvinte cheie: diabet zaharat tip 2, nefropatie diabetică, proteina C reactivă, albumină urinară

2010.20.2(66)  Fiziologia - Physiology

19

AUTOIMMUNE ENDOCRINE ASSOCIATIONS IN ADULTPATIENTS WITH AFFECTED GLYCEMIC METABOLISM AND
THYROID DISORDERSADRIANA GHERBON1, ROMULUS TIMAR2, IOANA ZOSIN3
1
Department of Physiology, UMF “Victor Babeş” Timişoara
2
Department of Internal Medicine III, County Hospital Timişoara
3
Department of Endocrinology, County Hospital Timişoara

ABSTRACT
Autoimmune pathology appears by almost all endocrine glands and affects them in a variable incidence. Type 1 diabetes associatedwith certain endocrine disorders, pictures the autoimmune polyglandular syndromes. The purpose of this study is to determine theprevalence of the combination of different kinds of autoimmune disorders in adult patients with deterioration of glycemic balance andthyroid diseases. The studied group was of 650 cases with an age between 17-79 years.
The group of adults was subdivided according to the type of changes in the glycemic balance in 4 subgroups: DM group with type 1
represented by 60 cases (9.23%), DM group with type 2 represented by 290 cases (44.61%), a group with IGT represented by 183
cases (28.15%) and a IFG group of 117 cases (18%).
Keywords: diabetes mellitus, autoimmune thyroid disease, autoimmune polyglandular syndromes

INTRODUCTION
The autoimmune process is the most frequent cause of
endocrine disorders. Autoimmune thyroid diseases tend to associate among themselves or with other autoimmune disorders.
Polyglandular syndrome associates multiple autoimmune endocrine disorders with other nonendocrine disorders at the same
person and their families (19).
DM type 1 is commonly associated with endocrine disorders
and systemic autoimmune pathology such as: Graves-Basedow
disease, Hashimoto thyroiditis, Addison’s disease, Celiac disease, Pernicious anemia, Myasthenia gravis, Vitiligo. (5).
1 in 100 patients with DM type 1 will develop Graves’s
disease and 1 to 20 patients are affected by hypothyroidism(7). The incidence of type 1 DM association with hyperthyroidism and hypothyroidism varies from 3.2% to 4.6%, respectively
from 0.7% to 4% (15).
1 to 200 patients with type 1 DM (7) will develop Addison’s
disease which is extremely rare in the general population.
Celiac disease appears at 1 in 20 patients with type 1 DM,
Pernicious anemia in 1 of 50 patients with type 1 DM (7).
A particular combination of type 1 with DM and hypo-or hyperthyroidism is characteristic in the polyglandular autoimmune
syndromes. There are 3 autoimmune polyglandular syndromes.
The autoimmune polyglandular syndrome type 1 (PAS- 1) is an autosomal recessive disorder caused by a mutationin the short arm of chromosome 21, characterized by the triad:
muco-cutaneous candidiasis, hypoparathyroidism and Addison’s
disease.
The symptoms and signs appear in childhood; candidiasis
is usually the first sign, followed usually by hypoparathyroidism
and Addison disease (2. 14). DM type 1 occurs in less than 4%of affected children, but increases to 12% by adults.
Autoimmune polyglandular syndrome type 2 (PAS - 2)
(4. 17) is the most common endocrinopathy. Occurs in adultlife and affects mostly women. The same patient has two or
more of the following conditions: Addison’s disease, Graves’s
disease, autoimmune thyroiditis, DM type 1, primary hypogonadism, Myasthenia gravis and Celiac disease. Most disorders
are associated with the following HLA: A1, B8, DR3 (DQA1 *0501, DQB1 * 0201) and DR4 (DQA1 * 0301, DQB1 * 0302).
The autoimmune syndrome disorders present usually a longprodromal phase and the antibodies are present prior to thedevelopment of the disorder.
Autoimmune polyglandular syndrome type 3 (PAS - 3)(3) is a PAS 2 syndrome, but without the adrenocortical involvement. It comprises a group of autoimmune disorders characterized by severe glandular insuffiency. A quarter of the patientswith hypo functional glands present other endocrine diseases
as well. This syndrome is associated with diseases as: celiac
disease, hypogonadism, myasthenia gravis, sarcoidosis, Sjogren
syndrome, rheumatoid arthritis, gastric neoplasia, malabsorption,and may be classified in 3 subcategories:- PAS III A - autoimmune thyroiditis with DM type 1- PAS III B - autoimmune thyroiditis with pernicious anemia- PAS III C - autoimmune thyroiditis with vitiligo and / oralopecia and / or other autoimmune diseases
DM is associated although with other endocrine disorders
and autoimmune polyglandular syndromes (6) faced with a much
lower frequency.
Insulin resistance Syndrome Type B is caused by thepresence of the insulin antibodies. A third of these patients haveassociated autoimmune disorders, as systemic lupus erythe-

Received February 2010. Accepted May 2010. Address for correspondence: Adriana Gherbon, Md, PhD, Physiology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, 2A Eftimie
Murgu Square, RO-300041, Phone/Fax: +40256490507, e-mail: adrianagherbon@yahoo.com

20

Fiziologia - Physiology  2010.20.2 (66)

toimmune thyroiditis with pernicious anemia
utoimmune thyroiditis with vitiligo and / or alopecia and / or other autoimmune

20 – 29
30 – 39
40 – 49
50 – 59
60 – 69
70 – 79

29
48
168
219
118
57

4.46
7.38
25.84
33.7
18.15
8.77

27
43
141
209
112
46

93.1
89.58
83.93
95.43
94.91
80.7

2
5
27
10
6
11

6.9
10.42
16.07
4.57
5.09
19.3

Case prevalence

Case prevalence

iated although with other endocrine disorders and autoimmune polyglandular
with a much lower frequency.
ance Syndrome Type B is caused by the presence of the insulin antibodies. A third
50%
44,61%
autoimmune disorders,
thyroid pathology.
spite of elevated50%
e associatedmatosus,
autoimmune
asInsystemic
lupus erythematosus,
autoimmune
44,61%
levels of plasma
glucoseof
secondaryto theglucose
insulin resistance,
40% to 40%
spite of elevatedlevelsplasmasecondary
the insulin resistance,cetoacidosis is not characteristic. Patients may present sponta28,15%
28,15%
30%
30% and severe hypoglycemia
aracteristic.neousPatients
present
spontaneous
remission
remissionmay
and severe
hypoglycemia(secondary effects
18%
18%
insulin-like antibodies,effects
demonstrated in vitro).
nsulin-likeofantibodies,effects
demonstrated
in vitro). 20% 20% 9,23%
10%
Poems
syndrome
includesDM,
primary
hypogonadism,
sen9,23%ome includes DM, primary hypogonadism, sensitive 10%
and motor
neuropathy, bone0%
sitive and motor neuropathy, bone lesions, hyper pigmentation.
DM type 1
DM type 2
IGT
IFG
tation.
DIDMOAD syndrome is an autozomal recessive disorder,
0% Fig.1. The allocation of cases based on changes in the levels of plasma
diabetes insipidus,
DM, opticdisorder,
atrophy, deafness.
yndrome which
is anincludes
autozomal
recessive
which includes
diabetes insipidus,
DM type 1
DM type 2
IGT
IF
glucoseDiabetes
mellitusisusuallythefirstmanifestationinchildren.
afness. Diabetes mellitus is usually the first manifestation in children.
Down syndrome is relatively common associated with
METHODSINVESTIGATION
me is relatively
commonassociatedwithpresenceDM
typeof1OF
andthyroiditis.
Fig.1.
The ofallocation
cases
based on changes in the levels of pl
the presenceof DM type
1 and thyroiditis.
Thisthesuggeststhatromosomalchromosomal
abnormalities
affectprocessesor that the susceptibility toabnormalities
affectautoimmune
autoimmune processesor
The methods of investigation were represented by clinicalthat bethe susceptibility
to develop
diseasesdisorders.
may bediseases may
associatedwith autoimmune
chromosomaldata- case history, current status, imagistic- thyroid ultrasonograassociatedwith chromosomal disorders.
METHODSOF INVESTIGATION
of autoimmune
disordersisimportanttodetermine
for
each
gland
their specificphy,biochemical- carbohydrate
metabolism parameters: fasting
For diagnosis of autoimmune disorders is important to deterplasma
glucose,glycosuria,
HbA1;
thyroid hormones and some
antibodies mine
areforrepresented
by ICA,
GAD,
IA2 for DM type 1, the antithyroid
each gland their specificantibodies.
Specific antibodies
immunologicalparameters.
The
methods
investigation were represented by clinical data - c
represented
by ICA,GAD, IA2
for(20).
DM type 1,of
the antithyroid
H antibodiesarefor
Addison's
diseaseDetermination of plasma glucose was performed by enzyme
antibodies,
and
21-OH
antibodies
for
Addison’s
disease(20).
imagisticthyroiddisorderultrasonography,
biochemical- carbohydrate
metabolism
gnosis of an autoimmune
endocrinethepatient
shouldbeglucosooxidasis.
investigatedforvalues were taken
techniquewith
NormalbeIn case of diagnosis of an autoimmune endocrine disorder
glucose,glycosuria,
HbA1;
thyroidhormonesandsomeimmunologicalparame
tween
70
110
mg%;
diabetes
mellitusvalues
over
126
mg%,
ders that canbe inshould
thebelatent
phase.
secondstep is to find specific biologicalthe patient
investigatedfor otherTheendocrine
disorders
impaired glucose
tolerance- values betweenby
110 - enzyme
126 mg%plasma glucose
wasperformed
technithat can be in the latentDetermination
phase. The second step is toof
find specific
and.
and
the
OGTT
at
2
h
between
140
200
mg%
and
impaired
Normalvaluesbiologicalparameters foreach gland.were taken between 70 - 110 mg%; diabetes mellitus - value
fasting glucose - values between 110 - 126 mg% and OGTT at
glucose tolerance - values between2 h110
- 126
under 140
mg%. mg% and the OGTT at 2 h be
METHOD
impaired fasting glucose - values between
110
- 126
Determination of HbA1c
wasmg%
achieved andthroughOGTT
the DiaStat at 2 h un
for measuring
HbA1c reported
to the total
HbA.
Determination of HbA1c wasachievedthroughthe
DiaStat for measu
INVESTIGATED POPULATIONOPULATION
To
determine
the
TSH
level
in
plasma,thefree fraction of
The totalgroup ofHbA.
subjects included 650 people, young adults,
triiodotironin(FT3),
andtheplasmafree
fractionofthyroxin (FT4)
ubjects included
650
people,young
adults,adultsandelderlypeople,aged
betweenadults and elderly people,
between 17 andthe
79 years(Tab. level in plasma, the free fraction
ToageddetermineTSH
were
performedaquantitative
methodARCHITECT;
witchofis antriiodotiro
I). The subjects
were withdiabetes andand inintimedevelopedI). The subjects
were
with
diabetestimedeveloped
thyroiddisordersaswellfree fraction of thyroxin (FT4) immunologicalwere performed
a quantitative
method A
method, (CMIA) Chemilumnescent
Microparticle
thyroid disorders as well or subjects with thyroid disorders who
oid disorders
whodeveloped in time
anomaliesin the processof glycoreglation
or TSH = 0465-4.68Immunoa
Immunoassay.
Normalvalueswere
following:
immunologicalmethod,(CMIA)
Chemilumnescent
Microparticle
developed in time anomalies in the process of glycoreglation or/ ml,FT3
FT3 = 3.69
-10.4 pmol
/ l, FT4 pmol
= 10-28.2 /pmoll.
diabetesfollowing:
mellitus.
TSH = 0465-4.68 Miu / Miu
ml,= 3.69-10.4
l, /FT4= 10-28
To obtain the level of cortisol was performed the technique
To obtain the level of cortisol
wasperformed
technique
IMMULITE/ IMMULITE
1000, anthe
imunometricmethod, inIMMULI
Table I. Distribution by age and sex in the group of adults
Table I. Distribution
by
age
and
sex
in
the
group
of
adultsimunometric method, in solid phase,competitive,of chemiluminescent,ICEsolid phase,competitive, of chemiluminescent,
ICEM (Immuno
Age Enzymometric
Number ofSex F
M considered
Chemilumino
Enzymometricassay).5-25
It was considered
normal: / dl.
assay).
It Sexwas
normal:a.m.
microgramcases
a.m. 5-25 microgram / dl.
FSHquantitatively by the ARCHITECT men%nlevel% wasn measured%
FSH level was measured quantitatively by the ARCHITECT
Microparticle
Immunoassay.
Reference
determinedwith ARCHITECT
method; avalues:
Chemilumnescent
Microparticle Immunoassay.
Refer18 – 19
11
1.7
10
90.9
1
9.1
ence values: determined with ARCHITECT test.

Table II. The reference values for FSH
Table II. The reference values for FSH

Population fieldmIU/mlWomen:- Follicular phase
3.35 – 21.63
The group of subjects was subdivided according to the- Ovulating phase
4.97 – 20.82- Luteal phaseof change in theaccording
glycemic balance
4 subgroups
1):
ubjects wastypesubdivided
to inthetype of(fig.
changein the glycemicbalance in1.11
4 – 13.99
Postmenopausal
2.58– 150.53-group DM type 1 represented by 60 cases (9.23%)-group DM type 2 represented by 290 cases (44.61%)

1.37 – 13.58
Men
presented by-group
60 cases(9.23%) by 183 cases (28.15%)
IGT represented
presented by-group
290IFGcasesof 117 (44.61%)
cases (18%)
The immunological parameters were represented by auThe immunological parameters
were represented by autoimmune thy
ted by 183 cases (28.15%)
toimmune thyroid markers - antibodies (antiTPO and antiTg(antiTPO and antiTg antibodies). antibodies).
ases (18%)
To determine serum levels of antiTPO antibodies it was used the kit
method (Microparticle Enzyme Immunoassay). Normal values: antiTPO
antib
21
2010.20.2(66)  Fiziologia - Physiology
To determine serum levels of antiTg antibodies it was used the kit
method as well (Microparticle Enzyme Immunoassay). Normal values:AntiTg
To determine 21-hidroxilaza (anti 21-OH antibodies) antibodies
radioimunodetermination method combined with a technique of imunoprecipi

DM tip 1 + TCA + vitiligo
DM tip 1+ TCA + Addison diseaseDM tip 1 + TCA + autoimmune ovarian failure
DM tip 1 + Graves-Basedow disease + vitiligo
TCA + Addison disease
PR + Graves –Basedow disease
TCA + vitiligo
Graves-Basedow disease + vitiligo
DM tip 1 + TCA + vitiligo + Biermer anemia

3
7
6
1
4
2
3
1
1

0.46
1.07
0.92
0.15
0.61
0.3
0.46
0.15
0.15

To determine serum levels of antiTPO antibodies it was
In the group of adults the main endocrine autoimmune asused the kit AxSYM an immunological method (Microparticlesociations are represented in Tab. IV:
the group
adults the main endocrine autoimmune associations are repres
Enzyme Immunoassay). NormalIn
values:antiTPO of
antibodies
Table IV. Prevalence of the main endocrine autoimmune associations in the(<35 IU / ml).
studied group
To determine serum levels of antiTg antibodies
it was
used
the
Table
IV.
Prevalenceof the main endocrine
autoimmune associations in the studie
kit AxSYM antiTg, a MEIA method as well (Microparticle Enzyme
Endocrine autoimmune associations
No.
%
Immunoassay). Normal values:AntiTg antibodies (<55 IU / ml).
DM tip 1 + TCA
37
5.69
To determine 21-hidroxilaza (anti 21-OH antibodies) antibodies
DM
tip
1
+
Graves–Basedow
disease6
0.92
level it was used the radioimunodetermination method combined
DM tip 1 + TCA + autoimmune ovarian 6
0.92with a technique of imunoprecipitation, based on human 21-OH
failuremarked with I 125 reacting with the antibodies anti 21-OH from theDM tip 1 + TCA + Addison disease
1.97
25 reactingsamples
withtestthe
antibodies
anti that
21-OH
from
test and forming 74
and forming
immune complexes
precipitatedwith the samples
TCA + Addison disease
0.61
the solid-phaseof protein
A. Normal range:
IU / ml A. Normal range: <1 IU / mlat precipitatedwith thesolid-phaseof <1proteinThyroid ultrasonography was performed in all cases andonography allowed
was performed
in allvolume,
andstudy
allowedto measure
volume,InDM
the groupof thyroid
adultswithfirst
DM
type1
the first imunopathy
wasDM type 1
Incasesthethyroid
group
ofandadults
type1
the
imunopathy
was
us to measure thyroid
the uswith
DM
type
1,
present
in
33
of
the
cases.
TCA
was
associatedinchanges inparenchyma’s
density.
Andensity,
increased
density,uniform,characterizes
changesin parenchyma’s
density.
An increased
uniform, incases.
TCA
was
associated3131cases
andBasedow
diseasein4 cases,
2 cases.
In 4 cases,
cases
and
Basedow
diseasein2
cases.
In
thyroidcharacterizes
normal thyroid
parenchyma
easilydistinguishedhyma easily
distinguished
from
the
neck
muscles
that
are
hypo
dens.
DM type 1 were detected at the samewith TCA
andtime2 (2cases
disordertimeand DM(2typecases
1 were detected
at the samecases of Gravesfrom the neck muscles that are hypo dens.
with
TCA
and
2
cases
of
Graves-Basedow
disease).
In
19
casesand 2 caseprocesses andInflammatory
autoimmune
pathology
appears
hypo
dens.
The
scale
was
assessedas
19 casesthyroid disorderpreceded the DM type 1 (17 cases with TCA
processes
and autoimmune
pathology apthyroid
disorderpreceded
the
DM
type
1
(17
cases
with
TCA
and
erate ++ andmarked+++.
pears
hypo dens.
Thedisease)scale was assessed
as being discreet +,(Tab. V).
2
cases
of
Graves-Basedow
disease)(Tab.
V).
moderate++ andmarked+++.
mune thyroid
diseasetheparenchymaof theappearswith
hypo
dens.
To track
theglandassociation
Addisondiseaseit wasdisease
determined
To track
the association
with Addison
it was deter- the plasma
In the autoimmune thyroid disease the parenchyma of the
mined
the
plasmalevelofcortisoland21-OH
antibodies
by 39
isease is seenanincreasedvolumeofthethyroid,hypoecogenityofdifferent
21-OH
antibodies
by39
adultstaken
inthestudy.
The
valuesof
21-OH
antibodi
gland appears hypo dens.
adultstaken
inthestudy.
The
valuesof
21-OH
antibodies
were
le homogeneity
in thedisease
thyroidparenchyma.
In Graves’s
is seenanin
increasedvolume of theincreased
7 cases.
significantly increased in 7 cases.
thyroid,hypoecogenityofdifferent
intensities
withvariable
mmune thyroid disorder appears
with athehypoecogenityof theand insufficiency
Tracking
association
withautoimmune
ovarianTracking
the parenchyma
association with
autoimmune
ovarian insuffi- led to dehomogeneity in the thyroid parenchyma.
ciencylevels of FSH,
which wasinsufficiency
increasedyroid volume.Chronic autoimmune
FSH,
which
increased/ lledinto 6determine
cases.thePrimaryovarian(earl
thyroiddisorderwas
appears
with a hypo-> 25 IU
>
25
IU
/
l
in
6
cases.
Primary
ovarianinsufficiency(earlymenoecogenity of the parenchyma and normal or increased thyroid
pause) usually occurs before the age of 40 years (in the absence
CUSSIONvolume.
of iatrogenic causes) and it s clinical signs are secondary amenorrhea and hypergonadotropism with hypoestrogenemia.
RESULTS AND DISCUSSION
Autoimmune ovarian insufficiency (AOI) is usually associatedadults 9.23% had type 1 DM, 44.61% had DM type 2, with
IFG
28.15% and 18% ITG.
other autoimmune pathology such as diabetes mellitus type
In
the
group
of
adults
9.23%
had
type
1
DM,
44.61%
had
immune combinations were represented by DM type
1, disease,
Addison's
disease,1, Addison’s
TCA, vitiligo,
etc.; its diagnosis is difficult
DM type 2, IFG 28.15% and 18% ITG. The main endocrine imanditisusually
based
on
the
exclusionof other possible causes
disorder (TCA),
autoimmune
ovarianinsufficiency
andthenonendocrine
mainmune combinations were represented by DM type 1, Addison’sofprimary
ovarianinsufficiency
andthenotice of autoimmune
disease, autoimmune
thyroid disorder(TCA), autoimmune
itiligo, alopecia,
Biermeranemia,rheumatoidetiologyarthritis(Tab. III)(3).
ovarian insufficiency and the nonendocrine main disorders were
In our group of subjects the patients had organ-specificvitiligo, alopecia, Biermer anemia, rheumatoid arthritis (Tab. III)autoimmune endocrine disorders shown in Fig. 2.
Table III. Prevalenceof endocrineautoimmune
autoimmune disorders
in the studiedin
groupable III. Prevalenceof endocrine
disorders
the studied Endocrinegroup immunopathys may be linked to a variable incidence of systemic organ-specific nonendocrine disorders.
Associations

Subject
group
No.
%

DM tip 1 + TCA
DM tip 1 + Graves-Basedow diseaseDM tip 1 + TCA + decalvant pelad
DM tip 1 + TCA + vitiligo
DM tip 1+ TCA + Addison diseaseDM tip 1 + TCA + autoimmune ovarian failure
DM tip 1 + Graves-Basedow disease + vitiligo
TCA + Addison disease
PR + Graves –Basedow disease
TCA + vitiligo
Graves-Basedow disease + vitiligo
DM tip 1 + TCA + vitiligo + Biermer anemia

31
5
2
3
7
6
1
4
2
3
1
1

4.76
0.76
0.3
0.46
1.07
0.92
0.15
0.61
0.3
0.46
0.15
0.15

AOI

10%

ADDISON

1,38%

B ASEDOW

10%
83,33%TCA
DM type1

93,33%
0%

20%

40%

60%

80%

100%

Case prevalenceFig 2. Endocrine imunopathys distributed on the number of cases

 2010.20.2 (66)
adults the 22
main endocrine autoimmune associations are representedFiziologia
in Tab.- Physiology
IV:

V. Prevalence of the main endocrine autoimmune associations in the studied group
Endocrine autoimmune associations

No.

%DM type1

93,33%
0%

20%

40%

60%

80%

100%

Case prevalenceFig 2. Endocrine imunopathys distributed on the number of cases

8 cases it was
associatedvitiligo, whichbefore
occurred before
with type 2 DMimunopathys.
PAS type 3 C was found in 2 (0.69%)
associated Invitiligo,
whichoccurredthe onset Inofadults
endocrine
the onset of endocrine imunopathys. Pelade decalvant appearedcases, of which 1 case (50%) Graves Basedow disease + rheured in 2 incases
andalsoalso
precededthe onset
2 cases andprecededthe onset of autoimmune
endo- of autoimmune
matoid arthritis and 1 endocrinopathys.
case (50%) TCA + vitiligo. Both cases were
Rheumatoid
arthritis inappeared
one case.ofOne
median age was 55 ±endocrine
14.14 years and the average
peared in crinopathys.
one case.
One case
the ingroup
adultsfemales.
withTheautoimmune
case in the group of adults with autoimmune endocrine diseases
age of onset was 53.5 ± 12.02 years. It was no thyroid familial
more thanhadone
nonendocrin autoimmune disorder history
respectively
Biermer anemia
associated more than one nonendocrin autoimmune disorderdisease found.
respectively Biermer anemia and vitiligo.

PAS type 2 was found in 4 cases (1.34%) TCA + Addison
disease. The average interval of time between the onset of AdTable V. Range (years) between the onsets of imunopathys in adults with type
dison disease and TCA was 16 years. All 4 cases were females.
Range (years) between the onsets
of imunopathys in adults withTheirtype
1 diabetes
1 diabetes
averageage was 61.75 ± 7.32 years and the mean ageof onset of thyroid disease was 60.75 ± 8.38 years. It was no
Period of time
No.
Media ± SD
thyroid familial history disease found (Fig.4).
of
(age)
cases

Onset DM – Onset TCA
Onset TCA - Onset DM
Onset DM – Onset Basedow
Onset Basedow - Onset DM
Onset Addison – Onset TCA

31
17
2
2
4

22.29 ± 12.42
2.47 ± 1.94
9.5 ± 4.94
5.5 ± 6.36
16

1%
1%
1%

1%

In adults, the average time between the onset of type 1 DM
1%and immunopathys was 22.29 ± 12.42 years. In 17 cases the firstrage timeimmunopathy
betweenwasthe
onset
of
type
1
DM
and
immunopathys
was 22.29 ± 12.42
TCA, followed at a distance of 2.47 ± 1.94 years0%
rst immunopathy
wasTCA, followed
atwerea initiallydistance of 2.47
± 1.94 years by DM
by DM type 1. By
Basedow-Graves
disease: 2 cases0%with DM,
followed atwerea distanceof 9.5 ± 4.94
yearsDM,of Gravesaves disease:
2 cases
initiallywithfollowed at 0%a distance of 9.5 ± 4.94
Basedow disease and 2 cases were initially with Basedow-Graves
TCA + ADDISON
PR + BASEDOWTCA + VITILIGO
w diseasedisease,and followed
2 caseswerewithdisease,followed
at
at intervalsof 5, initially
5 ± 6.36 years of
DM typeBasedow-Graves
1. By
1,34%
0,34%
0,34%
DM type2
Addisondiseaseassociatedthe first immunopathy
was associated with TCA the first36 years ofDMtype
1. withByTCAAddison
diseaseFig.4. Association of endocrine and non-endocrine immune diseases by
Addison’s disease followed at a distance of 16 years by TCA. In
adults withtype
DM type 11 DM
son's disease
followedatadistance
of
16
years
by
TCA.
In adults withadults with type 1 DM PAS type 3 has appeared in 49 (81.66%)
cases and PAS
type 2andin 7 (11.66%)
d in 49 (81.66%)
cases
PAScases.
type 2 in 7 (11.66%) cases.
In adults with IGT, PAS type 3 C was found in 2 cases
The average interval of time between the onset of DM anderval of time
between
the
onset
of
DM
and
thyroid
diseasewasTCA+
22.29(1.09%):
1 case (50%)
vitiligo±and12.42
1 case (50%) Graves
thyroid disease was 22.29 ± 12.42 years for TCA and 9.5 ± 4.94
Basedow
disease+rheumatoid
arthritis(Fig.5).
Graves Basedow
disease. disease.
4.94 yearsyears
forforGraves
Basedow
Both cases were females. The median age was 60 ± 4.24
The average interval of time between the onset of thyroid
rval of time
between
the
onset
of
thyroid
disease
and
the
type
years
andappearance
the average age of of
onsetDMof thyroid
disease was 59 ±disease and the appearance of DM type 1 was 2. 47 ± 1.94 years
2.82
years.
Itwasnothyroidfamilial
history
diseasefound.
for TCA and
5.5
±
6.36
years
for
GravesBasedow
disease.
for TCA and 5.5 ± 6.36 years for Graves Basedow disease.
60%

1%

50%

1%

40%

0%

30%

0%

20%

0%

10%

0%

0%

0%
DM type
1+TCA
DM type 1

DM type
1+DM

DM type DM1+vit DM1+T DM1+G DM1+T

type2+T 1+TCA iligo+alo CA+AO raves+vi CA+Add

Graves CA+alop +vitiligo

51,66% 8,33%

3,33%

5,00%

pecia
1,66%

I

tiligo10,00% 1,66%

TCA + VITILIGO

PR + BASEDOW

0,54%

0,54%

ison
11,66%

Fig.3.Association of endocrine and non-endocrine immune diseases by adults
with DM type 1

2010.20.2(66)  Fiziologia - Physiology

IFG

Fig.5. Association of endocrine and non-endocrine immune diseases by
adults with IGT

In adults with IFG, PAS type 3 C was found in 2 (1.7%) cases:23

1 case (50%) Graves’ disease + vitiligo and 1 case (50%) TCA+ vitiligo (Fig.6). Both cases were females. The median age
was 55 ± 5.65 years and the average age of onset of thyroid
disease was 51 ± 11.31 years. Thyroid familial history disease
was present in one case.

1%
1%
1%
1%
1%
0%
0%
0%
0%
0%
IFG

BASEDOW + VITILIGO

TCA + VITILIGO

0,85%

0,85%

Fig.6. Association of endocrine and non-endocrine immune diseases by
adults with IFG

PAS 1 is a very rare disorder. The largest number of patientswas reported in Finland, where the prevalence was estimatedat 1 in 25,000 subjects. The ratio is F / M 0.8 - 1.5 / 1, and in1998 the ratio was 2.4 / 1. The onset is in the childhood with
3 - 5 years old or in the early adolescence, but always before
the third decade of life. Death is determined by the individualcomponents of the syndrome. (2)PAS 2 is quite often in the U.S. about 14-20 to 1 million
people are affected by this syndrome. The ratio of F / M is 3 - 4/ 1 and the onset is in the third and fourth decade of life. So far,the mortality and morbidity rate hasn’t been assessed clinically,
but it is believed that it equals the mortality and morbidity rateby individual components (17).
The prevalence of PAS 3 is unknown. It is more often met inwomen than by men; usually by middle-aged women but it canoccur in people of any age. Death is determined by the individualcomponents of the syndrome (3).
In general, in the first stage of PAS antibodies levels areelevated. In the second stage the disease is sub clinical and in
the third stage becomes clinically manifested.
In the study group, all patients had type 1 DM clinicallymanifest, all being treated with insulin in different therapeutic
schemes. By patients with DM type 2, and IGT, IFG the treatment was diet.
If Graves - Basedow disease was associated, all had hyperthyroidism in treatment with antithyroid synthesis drugs.
By TCA, 112 cases were with euthyroidism and 96 caseswith hypothyroidism (76 clinical cases and 20 cases sub clinical). 36 cases did not require treatment; the remaining 172 hadsubstitution treatment with thyroid hormones. AntiTPO antibodies
24

were present by 27 of cases of TCA and DM type 1, a total of 23
cases presenting insignificant values. All 4 cases with Addison
disease were symptomatic and the diagnosis was on the basisof the clinical symptoms.
If Addison disease is associated the substitution treatment
can cause imbalance of DM, especially of type 1 DM (13).
Also estrogen therapy in autoimmune ovarian insufficiency
may increase the risk of cardiovascular disease (12).
The treatment with methotrexat used in rheumatoid arthritis,
proved to have toxic effects on liver and lungs (1).
Ideal is to determine the presence of antibodies, especiallyin DM type 1, because they may be present by subjects withoutclinical symptoms. If their levels are raised, it is good to monitor
annual the TSH level and if it is normal it is recommended to doze
antithyroid antibodies by intervals of 2-3 years (8,18).
Also, if the disease is autoimmune, the patient should be
investigated for other autoimmune associations of endocrine or
non-endocrine nature.
A study in Czech Republic on 51 patients with type 1 DM
showed that it is associated with autoimmune thyroid diseases,
with Addison’s disease and Celiac disease.
The authors recommend finding the specific antibodies foreach disease, to diagnose the disease in the initial phase, andto prevent the complications that will affect the quality of thepatients’ life (9).
If DM type 2 is present it is recommended to evaluate
TSH levels, and if it is normal, to repeat this evaluation every
5 years.
If pre-existing thyroid pathology is present it is recommended to evaluate plasma glucose levels annually.
CONCLUSIONSIn the lot of adults with various changes in the glycemicbalance and thyroid disorders, the main PAS encountered
was PAS 2 (1.68% cases), PAS 3 (7.5% cases) and PAS 3 C(0.91% cases) (Fig.7). The most common was PAS 3 A (7.5%)
because of the association of type 1 DM with TCA (both likely
autoimmune diseases).
0,91%

1,68%

7,5%

PAS 2

PAS 3aPAS 3cFig.7. Prevalence of PAS in the group of adults

Many disorders involved in PAS present a long prodromal
Fiziologia - Physiology  2010.20.2 (66)phase, characterized by the presence of characteristics antibodies for each disorder in part, before the clinical manifestations.
The treatment of patients with PAS involves early identification of all components.
The treatment of PAS is currently the treatment of eachcomponent of endocrine disorder (usually through hormone
substitution therapy). Iso-hormonal therapy has “immunomodulatory” capacities (hormone produced by the target organs may
be able to influence autoimmunity).
Associations of specific autoimmune endocrinopathys requires specific management:- substitution treatment with thyroxin may precipitate adrenal
insufficiency by untreated Addison’s disease- hypoglycemia or decreased insulin necessity in patientswith type 1 DM may signify the onset of a adrenal insufficiency- asymptomatic forms of Addison’s disease should be treatedcarefully under acute stress- in Addison’s disease associated with ovarian insufficiency,
substitution therapy with steroid hormones can prevent severe
osteoporosis- controversial discussions are described in the literature on
the effectiveness of thyroxin in patients with positive antibodies,but with euthyroidism or sub clinical hypothyroidism (10)Some show a significant reduction of the TSH and of theanti-TPO antibodies in patients with autoimmune thyroiditis andeuthyroidism after 1 year of treatment with thyroxin (10).
The PAS classification is not final. This may change overtime, with the onset of new endocrine disorders or associations
with new autoimmune determination. .
REFERENCES

1. Alarcon GS, Kremer JM, Macduso M, Weinblatt ME, Cannon GW,
Palmer WR, St. Clair EW, Sundy JS, Alexander RW, Smith GJW, Axiotis
CA. Risk Factors for Metrotrexate – Induced Lung Injury in Patients with
Rheumatoid Arthritis: A Multicenter Case-Control Study. Annals of Internal
Medicine, 1997; 127 (5): 356 - 364
2. Aldasouqi SA, Akinsoto OPA, Jabbour SA. Polyglandular Autoimmune
Syndrome, Type I, In Endocrinology (electronic book), 2006, pag. 1 – 18
3. Aung K, Salmon M. Polyglandular Autoimmune Syndrome, Type III, In
Endocrinology (electronic book), 2006, pag. 1 - 18
4. Betterle C, Lazzarotto F, Presotto F. Autoimmune polyglandular syn-drome Type 2: the tip of an iceberg?. Clin Exp Immunol, 2004; 137(2):225 – 33
5. Cooper GS, Stroehla BC. The epidemiology of autoimmune diseases. Autoimmun Rev, 2003; 2(3): 119 – 25
6. Dănciulescu R. Diabetul zaharat asociat bolilor endocrine. În: Tratat dediabet Paulescu, Editura Academiei Române, 2004: 429 – 438
7. De Block CE. Diabetes mellitus type 1 and associated organ-specificautoimmunity, Verh K Acad Geneeskd Belg, 2000; 62 (4): 285 - 328
8. Eisenbarth GS, Gottlieb PA. Autoimmune polyendocrine syndromes. N
Engl J Med, 2004; 350: 2068 – 2079
9. Gonem S, Wall A, De P. Routine screening for thyroid disease at patients with diabetes mellitus: is it worthwhile? Endocrine Abstracts, 2007;
13: P 317
10. Hossein GR, Tuttle M., Baskin HJ, Fish LH, Singer PA, McDermot MT.
Subclinical thyroid dysfunction: a joint statement on management from theAmerican Association of Clinical Endocrinologists, The American Thyroid
Association, and the Endocrine Society. J Clin Endocrinol Metab, 2005;
90: 581 – 585
11. Johnson JL. Diabetes Control in Thyroid Disease, Diabetes Spectrum,
2006; 19: 148 – 153
12. Kalantaridou SN, Braddock DT, Patronas NJ, Nelson LM. Treatment of
autoimmune premature ovarian failure.Case report.Human Reproduction,
1999; 14 (7): 1777 – 1782
13. McAulay V, Fier BM. Addison’s disease in type 1 diabetes presenting
with recurrent hypoglycemia. PostgradMed J, 2000; 76: 230 – 232
14. Myhre AG, Halonen M, Eskelin P. Autoimmune polyendocrine syndrome type 1 (APS I) in Norway. Clin Endocrinol (Oxf), 2001; 54(2): 211–7
15. Radaideh A, El-Khateeb M, Batieha AM, Nasser AS, Ajlouni KM. Thyroid function and thyroid autoimmunity in patients with type 1 diabetes mellitus, Saudi Med J, 2003; 24(4): 352 – 5
16. Reed SD, Sutton EL. Menopause: Premature Ovarian Failure, Womens Health, 2002
17. Sivarajah S, Fan CY, Akinsoto OPA. �����������������������������
Polyglandular Autoimmune Syndrome, Type II. In Endocrinology (electronic book), 2006
18. Tunbridge WMG, Vanderpump MPJ. Population screening for autoimmune thyroid disease. Endocrinol Metab Clin North Am, 2000; 29: 239– 253
19. White RD and Harris GD. “Birds of a Feather Flock Together”: Type
1A Diabetes and Other Autoimmune Disease States.Clin. Diabetes, 2006;
24 (1): 40 – 43
Winter WE, Harris N, Schatz D. Immunological markers in the diagnosis
and prediction of autoimmune type 1a diabetes. Clinical Diabetes, 2002;
20: 183 -191

ASOCIERI ENDOCRINE AUTOIMUNE ÎNTÂLNITE LA PACIENŢII ADULŢI CU
ALTERĂRI ALE ECHILIBRULUI GLICEMIC ŞI AFECŢIUNI TIROIDIENEREZUMAT
Procesele autoimune interesează aproape toate glandele endocrine, care sunt afectate într-o incidenţă variabilă. Diabetul zaharat
tip 1 asociat cu anumite endocrinopatii realizează tabloul poliendocrinopatiilor autoimune. Scopul acestui studiu este de a stabiliprevalenţa asocierii a diferitelor afecţiuni de natură autoimună la pacienţii adulţi cu alterări ale echilibrului glicemic şi afecţiuni tiroidiene. Lotul general studiat a fost reprezentat de 650 cazuri, cu vârste cuprinse între 17-79 ani.
Lotul de adulţi a fost subîmpărţit în funcţie de tipul modificării echilibrului glicemic în 4 subloturi: lotul cu diabet zaharat tip 1 reprezentat de 60 cazuri (9,23%), lotul cu diabet zaharat tip 2 reprezentat de 290 cazuri (44,61%), lotul cu scăderea toleranţei la glucoză
reprezentat de 183 cazuri (28,15%) şi lotul cu scăderea toleranţei la glucoză a jeun reprezentat de 117 cazuri (18%).
Cuvinte cheie: diabet zaharat, boala autoimună tiroidiană, poliendocrinopatie autoimună

2010.20.2(66)  Fiziologia - Physiology

25

LONG-TERM IMPLICATIONS OF CARDIOVASCULAR ANDRESPIRATORY MEDICATIONS IN ELDERLY ASTHMATICSSIMONA MACESANU, ELENA ARDELEAN
Pneumology Ambulatory Care, Emergency County Hospital, Timisoara“Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
Asthma in the elderly, but also in other age groups, shows a high incidence in last years. Frequent co-morbidities, especially in
cardiovascular pathology, in the elderly, require combined treatment, often it has side effects or contraindication, for other diseases
associated.
Often remove from treatment the asthma medication or the other an important drug from cardiovascular medication as a precaution
or unjustified reasons, produce from exhaustive policy of selling drugs and sometimes unjustified targets in our country. The goal of
the present paper is to raise and to define these issues, to demonstrate that we need medication every disease that is separately,
considering that the side effects or the existing contraindications, without forgetting the treatment of asthma in the elderly, main
target for elderly patients being maintaining a good quality of life.
Key words: cardiovascular medication, asthma, elderly

INTRODUCTION
Asthma is one of the most common chronic diseases in
the world, and WHO estimated that prevalence is increase. In
Romania asthma prevalence is around 7-8% in the population,that is over one million people is asthmatic (1).
Late-onset asthma (in patients over 50) is more common
than previously thought, and its prevalence rate is constantly
increasing to around 5% in the population.
Currently asthma in patients over 65 years creates additional problems in terms of diagnosis, treatment, chronic
disease monitoring, comorbidities, and additional costs of
disease and changes over time. In the patients with undiagnosed asthma, symptoms are not perceived as being similar
to those of asthma, the elderly may develop atypical asthmatic
symptoms and nocturnal cough may be their only symptom. In
these patients, symptoms may be falsely assumed to belong toother diseases or to other trigger factors such as heart failure
or other cardiac distress, thyroid disease, old age, pollution,smoking, or reduced tolerance to effort, and in fact, thesemorbidities can coexist with asthma (2).
Nowadays, in Romania is a continuous increase in prevalence of cardiovascular diseases, especially hypertension,coronary heart disease, stroke and last but not least, heart
failure. The cardiovascular diseases represent the main causeof premature death in Europe and are a significant cause of
disability, significantly contributing to the rising cost of healthcare (3).
Frequent co-morbidities, especially in cardiovascularpathology, in the elderly, require combined treatment, often
it has side effects or contraindication, for other diseases associated.
Often remove from treatment the asthma medication or
the other an important drug from cardiovascular medication
as a precaution or unjustified reasons, produce from exhaustive policy of selling drugs and sometimes unjustified targets
in our country.
When cardiovascular diseases and asthma coexist,
medications to treat cardiovascular disorders may cause an
exacerbation of asthma and viceversa.
Asthma in the elderly may either be due to late onset (over65 years) disease, or part of long-standing asthma which had
persisted into old age, while elderly asthmatics had significantly
more near-fatal episodes. The factors, more prevalent in theelderly, which predispose asthmatics to near fatal attacks are:1) delay in diagnosis and treatment,2) poor cardiorespiratory
reserve, 3) impaired perception of increasing airways obstruction, 4) blunted hypoxic ventilatory drives, 5) psycho-social andcognitive problems (4).
MATERIALS AND METHODS
A. Cardiovascular medications and their implications forcomorbid respiratory diseases

Received May 2010. Accepted June 2010. Address for correspondence: S.Macesanu, MD, Ambulatory Care, Emergency County Hospital, Timisoara, e-mail: macesanusimona@yahoo.com

26

Fiziologia - Physiology  2010.20.2 (66)

A.1. Betablockers
The effectiveness of beta-blockers has been verified not
only for common indications (coronary heart disease, hypertension and certain forms of heart failure), but also in arrhythmias,
pheochromocytoma, dissecting aneurysm, portal hypertension,
hyperkinetic syndrome, glaucoma (in local administration).
Propranolol, one of the most frequently used antiarrhythmic
in the recent past, is now being replaced by selective antiarrythmics as metoprolol, betaxolol, nevibolol, but caution isrecommended when prescribed (5).
Antiarrhythmic sotalol is a broad spectrum drug, useful forvarious supraventricular and ventricular arrhythmias. Sotalol
may increase susceptibility to allergens and severity of anaphylactic reactions due to its effect of blocking beta-adrenergic
receptors. In patients with a history of hypersensitivity reactions
or in those under desensitization treatment there are high risksof severe anaphylactic reaction (6). The findings of SENIORS
study showed superiority of nebivololum (other commonly usedbeta-blocker) compared to placebo in reducing mortality andhospitalization rates in elderly patients with heart failure (7).
For patients with obstructive airway disease and concomitant congestive heart failure or ischemic heart disease
it is common practice to give ß-2 agonists while withholding
ß- blockers. There are now several case-control studies thathave documented an association between ß-2 agonist use andthe development of congestive heart failure, acute myocardial
infarction, or cardiac death. This association may be explainedby ß-agonist stimulation causing tachycardia, hypokalemia, andarrhythmia, especially in the presence of cardiac comorbidities
or hypoxemia. Cardioselective β-blockers do not produce clinically significant adverse respiratory effects in patients with mild
to moderate reactive airway disease. The results were similar
for patients with concomitant chronic airways obstruction (8).
A.2.Other antiarrhythmic medication
Amiodarone has the following side effects when taken on
a long-term basis: development of corneal pigment deposits,
hypothyroidism and diffuse pulmonary fibrosis.
A.3.Calcium channel blockers
Verapamil is an antiarrhythmic and bradicardizant
and produces vasodilation, and it is indicated in coronary patients with supraventricular arrhythmias because it slows theventricular rhythm and in junctional tachycardia. Verapamil has
numerous side effects, it exerts negative inotropism in heart
and can cause cardiac decompensation (9). It should be cautiously coadministered with Propranolol (because summationof myocardial depression can occur), Digitalis (there is a highrisk of heart block), Diltiazem it has verapamil-like properties,
and is used as antiarrhythmic and as antianginal treatment.
A.4.Cardiovascular medication that might induce or aggravate asthma or chronic cough in elderly patients (Table I)(10).

2010.20.2(66)  Fiziologia - Physiology

Table I. The influence long term of cardiovascular drugs in chronic obstructive
Table I. The influence long term ofrespiratory
cardiovascular drugs
in chronic obstructive respiratory diseases
diseases
Medication

Comorbidities at
Side effects
risk of being
treatedBeta-adrenergicHypertension
Asthma exacerbation
β blockers
Tremors
Bronchospasm
Glaucoma
Reduced responsiveness tobronchodilators
Non steroidal anti- Arthritis
Asthma exacerbationinflammatory drugs Musculoskeletal
Bronchospasm(NSAIDs)
disorders
Aspirin, fenofibrat may
cause pulmonary
eosinophilia
Non–potassiumsparing diuretics

Hypertension
Cardiacinsufficiency

Anticholinergic agents Urinary retention(ipratropium bromide, Glaucoma
tiotropium bromide)
ACE Inhibitors
Cardiacinsufficiency
Hypertension
Fenofibrat,amiodarone,
metisergida, acebutolol
Procainamide,
metildopamina,
quinidine, metisergida,hidralazine, phenytoin
Hidralazine, quinidine,
phenothiazines

Comments
To avoid as much as possible;
When recommended selective β
blockers will be chosen
Though not all elderly
asthmatics develop intolerance
to NSAIDs these drugs shouldstill be avoided as much as
possible

Worsening of cardiac Produce additive effects infunction / arrhythmias combination with asthma
induced by hypokalemia
medication:- ß2-agonists have anadditive effect with oral or
injected steroids and may cause
potassium loss
-coadministration with cardiac
glycosides
may causehypokalemia
Bronchospasm
Bronchorrhea

Ephedrine-based
medication
may aggravate urinary retention
and glaucoma

Increase in cough incidenceInterstitial pneumonitis,
alveolitis, pulmonary
fibrosis
Disseminated lupus
erythematosus withpulmonary determinations

In the long administration

Systemic polyarteritis withlung determinations

In the long administration

In the long administration

A.5.ACE inhibitors and statines
A.5.ACE inhibitors
statines
Angiotensin-converting
enzymeandinhibitors(ACE) are widely used in the management ofcardiovasculardiseases and systemic hypertension.
are the(ACE)
most commonlyprescribed
Angiotensin-converting
enzymeStatins
inhibitors
are widely
usedlipidlowering
agentsand have provenof
benefitin primary and secondary
preventionof coronary hyperheart disease.
in themanagement
cardiovasculardiseasesand systemic
There is increasing awareness that the broader pharmacologic properties of ACE and statins encompass
tension.
Statinslocal
arefibroproliferative
the most commonly
the abilities
to modulate
pathways in a prescribedvariety of organlipid-loweringsystems. In the lung,angiotensin
II isandemerging
a potentially
importantprofibroticand
mediator
via inductionof alveolar cellagentshaveasprovenbenefitin primary
secondary
prevention
apoptosis and as a fibroblast mitogen. Some of studies was demonstrates that ACE and/or statins are notofcoronary
heart
disease.
There
isincreasingawarenessthattheassociated with improved survival in IPF (11).
broader
pharmacologic
properties
of
ACE
and
statins
encompass
Patients who develop chronic cough in association with angiotensin-converting enzyme inhibitor
therapy
be switchedto an agent fromanotherdrug class. If coughpathways
persists, a chest
theshould
abilities
to modulate
local
fibroproliferative
inradiographa vari- andother investigations should be ordered to rule out malignancy and other serious conditions. ACE
ety
of
organ
systems.
In
the
lung,
angiotensin
IIisemerging
as ainhibitors cause a nonproductive cough in 5 to 20 percent of people (more often women than men). Thispotentially
importantprofibrotic
mediatorvia
inductionofalveolarside effect is not dose related, and the cough may begin one week to several months after ACE inhibitor
therapy
The coughin a fewmitogen.
days to several
weeks ofafterstudies
the ACE inhibitor
cellis initiated.
apoptosis
andshould
as asubside
fibroblastSome
was isstopped (12).

demonstrates that ACE and/or statins are not associated withimproved
survival
IPF (11).
B.Coexisting
asthmaand heartindiseasethe treatment implications of asthma in the elderly
Asthmamedications
may have increased
effects
in elderly patients
coexisting
Patients
who develop
chronic side
coughin association
withwithangiocardiovascular disease, so a treatment plan should be adjusted. Response to bronchodilator medication
tensin-converting enzyme inhibitor therapy should be switched to anagent from another drug class. If cough persists, a chest radiographand other investigations should be ordered to rule out malignancy
and other serious conditions. ACE inhibitors cause a nonproductive
cough in 5 to 20 percent of people (more often women than men).
This side effect is not dose related, and the cough may begin one
week to several months after ACE inhibitor therapy is initiated. The
cough should subside in a few days to several weeks after the ACE
inhibitor is stopped (12).
B.Coexisting asthma and heart disease- the treatment���������
������������������
implications of asthma in the elderly
Asthma medications may have increased side effects in elderly
patients with coexisting cardiovascular disease, so a treatment plan27

should be adjusted. Response to bronchodilator medication may
change with age. Elderly patients, particularly those who have associated ischemic heart disease, may develop tremor or tachycardia
after beta-agonists or theophiline administration. Concomitant useof beta-agonists and anticholinergics is recommended in elderly
asthmatics only if cardiac function is closely monitored (13).
B.1. Methylxanthines (aminophylline, theophylline)
Theophylline(THY) has been used the management of asthma
for more than 50 years and remains a widely prescribed anti-asthmatic agent worldwide.
Theophylline cleareance may be reduced by concomitantdiseases prevalent in elderly patients, and , which further impair
clearance of this drug and have the potential to increase THY bloodlevels, age appears to be an important independent iatrogenic risk
factor in high doses administration.(Shannon and Lovejoy 1990).The
efficacy and toxicity of THY are closely related to the serum drugconcentration.In patients receiving theophylline as monotherapy
for chronic asthma, doses providing peak serum concentrations
between 10 and 20 µg per milliliter are the most likely to prevent
symptoms and decrease the need for rescue therapy. However,bronchodilatory, antiinflammatory, and immunomodulatory effects
occur for THY lower serum concentrations, which may be adequate
for elderly (14).
Methylaxanthines have a number of drug interactions which
require some precautions in their administration on elderly. Someof side effects may be present in up to 50 percent of patients whenserum concentrations of 10 to 20 µg per milliliter are rapidly attained,
but they are much less frequent when the initial dose is low and thedose is increased at intervals of no less than three days so that theseserum concentrations are reached gradually. For THY serum concentrations >35 mcg/mL, both the frequency, and severity of adverse
reactions are increased, while cardiac arrhythmias, hypotension,
hyperglycemia, and intractable seizures which can be lethal, brain
damage, peripheral vascular collapse, or death may occur during
treatment with theophylline (15). Shin et al., Vestal et al. showed that
total cleareance (bound plus unbound theophylline) in the elderly
was 33%less than in younger groups. These observations indicatethat both renal and metabolic THY elimination processes are lessactive in the elderly (16).
Theophylline is currently a less preferred option than inhaledcorticosteroids recommended as a second-line choice of controller
in management of patients with asthma at Step 2 of the Global Initiative for Asthma Guidelines (GINA) 2002 guidelines (17). Although
long-acting inhaled ß2-agonists are more effective as an add-on
therapy at Steps 3 and 4 of the GINA 2002 guidelines, theophylline isconsiderably less expensive and may be the only affordable add-on
treatment when the costs of medication are limiting (18).
B.2. β2-agonists therapy as bronchodilator medication inasthmaB.2.1.Rapid-acting inhaled ß2-agonists
This is the medication of choice for relief of bronchospasm
during acute exacerbations of asthma and for prevention of
28

exercise-induced bronchoconstriction. Rapid-acting inhaled ß2agonists should be used only when they are strongly required andat the lowest dose required. Increased use of rapid-acting inhaledß2-agonists is a warning of deterioration of asthma control andindicates the need to reasses treatment.
Adrenaline has been used in the treatment of asthma since
the beginning of the 20th century. While adrenaline is an effective
bronchodilator, it also causes anxiety, profound stimulation of theheart, increase of blood pressure and excessive stimulation of thebrain. These unwanted side effects led the researchers to search foran adrenaline analog that would retain the bronchodilating quality
without the cardiovascular and central nervous system liabilities.
A close analogue of adrenaline, isoprenaline is free from vascularside-effects, but may cause an increase in the force and rate ofcontraction of the heart.
Salbutamol was found to be both a highly selective and potent
ß2-adrenoceptor agonist, with an adequate metabolic stability thatmade it a highly effective bronchodilator than fenoterolum or terbutalina. The most common side effects of Salbutamol are cardiac
tachyarrhythmias, QT-interval prolongation, ST-segment depression, some caution is recommended in patients with heart disease,coronary heart disease, arrhythmias or hypertension (19).
Metaproterenol, although not entirely free of beta1-receptor
effects, also improved specificity relative to both epinephrine andisoproterenol and provided a somewhat longer duration of action.
Oral preparations can be useful in elderly patients unable
to administer inhaled therapy, but with a much higher frequency ofadverse effects, particularly tachycardia or elevated blood pressure.
B.2.2 Long-acting beta-agonists (LABA)
Long-acting beta-agonists (LABA) are recommended for partiallycontrolled or uncontrolled persistent asthma in adults and in theelderly, usually in combination with inhaled corticotherapy, dependingon the stage of severity according to GINA.
Pharmacological studies in vivo and in vitro show selectivity on
beta2-adrenergic receptor and its similarity with isoproterenol, which
has approximately equal effect on beta1-and beta2-adrenoreceptors.
Studies show that salmeterol is 50 times more selective than isoproterenol on beta2-adrenergic receptors that prevail in bronchialsmooth muscle and beta1-adrenergic receptors that predominate
in the smooth muscle of the heart (10-50% of beta2-receptors
are found in the heart muscle). The high selectivity of salmeterol
on beta2-adrenergic receptors does not exclude the possibility ofcardiac adverse effects.
Drug interactions are reported at concomitant use of betablockers that may decrease bronchodilating, and vasodilating effectsof beta-agonists such as salmeterol; concurrent administration withmethyldopa may increase pressor response, ECG changes andhypokalemia resulting from diuretics may worsen when coadministered with salmeterol. Long-acting beta-agonists are contraindicatedin patients with documented hypersensitivity, angina, tachycardia,
cardiac arrhythmias associated with tachycardia. Castle W. et al.
show in asthmatic patients who require regular bronchodilator
treatment, the overall incidence of serious events from all causes
Fiziologia - Physiology  2010.20.2 (66)

(4% for salmeterol versus 41% for salbutamol) was unremarkable,
the incidence of those suspected as being related to the drugs(1.19% versus 1.15 %) was low, and importantly there was noevidence of any previously unrecognised side effect of salmeterolor salbutamol (20).
B.2.3.Combination therapy (CSI and LABA)
The combination preparation fluticasone and salmeterol usedin the management of asthma and chronic obstructive pulmonary
disease (COPD).
Whilst the use of inhaled combination steroids and LABA, arerecommended in asthma guidelines for the resulting improved
symptom control, concerns have been raised that the use of longacting inhaled bronchodilators such as formoterol and salmeterol
may increase the risks of asthma-related deaths and this additionalrisk is not reduced with the additional use of inhaled steroids. The
Salmeterol Multicenter asthma research Trial (SMART study) that
was a multicentre, randomized double-blind, placebo-controlled,
observational study initiated in 1996, and finished in 2003, had
positive findings. The SMART study has concluded that salmeterolor other long-acting β2-agonists (LABA) do not increase the risk forasthma-related deaths but it found a higher incidence of death inAfrican Americans with salmeterol therapy compared to placebo(African Americans presented high incidence of asthma symptoms,
deterioration in baseline PEF, more emergency departments visits
and more frequent hospitalizations, these facts being reported prior
to their study participation). The authors speculated on possible
genetic causes, mentioning β-receptor polymorphisms (21).
The long-term use of inhaled formoterol and budesonide could
causes systemic adverse effects that include coetaneous allergic
reactions, severe shortness of breath, arterial hypertension, tachycardia or arrhythmia, may significantly prolong the QTc interval in adose-dependent and gene-dependent manner, induce hypokalemia
and raise glycemia levels, tremor, anxiety, increased incidence of respiratory infections, eye-related ailments (cataracts and glaucoma),and may decreased bone mineral density.
Co-administration of combination steroids and LABA with otherbeta-blockers (eg, Propranolol) may block the pulmonary effect of formoterol, in addition to producing severe bronchospasm in asthmaticpatients but no other drug interactions have been found between
budesonide and medication used to treat bronchial asthma. Thus,
budesonide and formoterol combination should not be administeredin association with beta-adrenergic antagonists (including those inophtalmic drops), except for well-justified cases.
B.3.Oral steroidal anti-inflammatory drugs
Long-term use of orally inhaled corticosteroids may affectnormal bone metabolism, resulting in loss of bone mineral density.The decrease in bone mineral density is dosage-dependent
especially when it is associated with the use of inhaled corticoids,
even though high doses or medium doses are not associatedwith potential for major adverse effects. The elderly are at highrisks if they also have pre-existing osteoporosis, changes in
estrogens level that affects calcium absorption, or if they havea sedentary lifestyle. Patients with asthma who are on long2010.20.2(66)  Fiziologia - Physiology

term systemic glucocorticosteroids in any form should receive
preventive treatment for osteoporosis. Intake of calcium, Dvitamin supplements and estrogens has additional therapeutic
enjoying osteodensitometry assessment should be performed
every six months.
Co-administration with digoxin may cause digitalis toxicity
secondary to hypokalemia, phenobarbital, phenytoin, and rifampin
may increase metabolism of glucocorticoids (consider increasing
maintenance dose) and it is useful to monitor hypokalemia withcoadministration of diuretics (22).
B.4. Inhaled corticosteroids
Inhaled corticosteroidal medication has no adverse reactions
or side effects in cardiac patients, due to the low systemic doseabsorbed through gastrointestinal tract. ���������������������������
Inhaled corticosteroid therapy both improve clinical control and reduce exacerbations. Some
recent studies have found that the use of inhaled corticosteroid may
increase the risk of developing cardiovascular diseases and the riskof death. ����������������������������������������������������������
Other numerous studies indicated that inhaled corticosteroid therapy is associated with no risk of developing cardiovasculardiseases and no risk of death (23).
B.5.Leukotriene modifiers:
Clinical studies have demonstrated that leukotriene modifiers
provide clinical benefit, reduce asthma symptoms including cough,and improve lung function in older adult patients but not to thesame extent as in the case of young patients with associated atopy.
Leukotriene modifiers are well tolerated and no cardiovascularadverse reactions after long-term therapy with montelukast havebeen reported.
B.6.Inhaled anticholinergic medication
Inhaled anticholinergics are rapid-acting bronchodilatorswith therapeutic role that are less effective reliever medications inasthma than rapid-acting inhaled ß2-agonists and are recognized
as an alternative bronchodilator for patients who experience major
adverse effects from rapid-acting inhaled ß2-agonists.
Tiotropium is a long-acting, anticholinergic bronchodilator
used in the management of COPD and his side effects are concentration-dependent. In the elderly plasma drug concentration
increases and reduces renal function, thus tiotropium bromide
should not be administered in patients with moderate-severe
renal insufficiency only if clinical benefits overweigh potential
risks. Long-term data on the efficacy of tiotropium administration in asthma patients with severe renal insufficiency is lacking.
The occurrence of adverse drug effects inhalation of tiotropium
can cause respiratory tract infections, tachyarrhythmias, urinary
retention.
B.7. Nonsedative antihistamines (terfenadine and astemizole)used in allergic rhinitis can produse worsening of cardiac function
or ventricular arrhythmias, induced by prolongation of QTc interval.
We found increase incidence asthma and allergic rhinitis in elderlyin our area, involving attention both disease diagnosis and treatment (24).
29

B.8. Inhaled aerosol terapy
According to the respiratory tract anatomy, pharmacokinetics
and dynamics of the flow characteristics of inhalation therapy wasrecommended as the best prevention and treatment of chronic
asthma treatment methods. Using inhaled corticosteroids steroids
aerosols(Budesonide), β2-aerosol receptor agonists (Salbutamolul
ampoules), aerosol anticholinergic drugs (ipratropium), have fewerside effects in elderly and do a better penetration of aerosol (25).
DISCUSSIONS AND CONCLUSION
Asthma management in the elderly follows the same national
and international guidelines for asthma treatment and control thatare used in young adults, additionally psycho-emotional and socialchanges associated with ageing should be also considered, addressed and treated as appropriate.
Elderly asthmatics have different clinical characteristics fromyouths and we are required to understand these differences.Bronchial asthma has more severe clinical forms in the elderly than in
adult or young patients and it is associated with significant morbidity
and mortality (26). Therapeutic management of bronchial asthma
in the elderly requires close attention to the person’s adherence/compliance to the medication plan, to achieve and maintain control
of asthma symptoms, to prevent exacerbations, main target for
elderly patients being maintaining a good quality of life.
Asthma treatment and attainment of control in the elderly arealso complicated by: refusal to use aerosol devices for asthma, lackof family support, poor medical knowledge, high costs of asthma
medications, denial of asthma diagnosis, the occurence of adverse
drug reaction that are not perceived by elderly patients, lack of confidence in the efficiency and necessity of asthma medications, poor
self-monitoring of athma treatment and control (27,28).
Bronchial asthma raises frequent problems in terms of differential diagnosis, and is associated with numerous comorbid conditions
that complicate the overall choice for a medication plan, it is also
associated with risks of polypragmasy, and with high probability ofadverse drug reactions.
The lack of asthma management in cases of uncontrolled asthma
with frequent exacerbations in order to protect the asthma patientswith cardiovascular co-morbidities against the risk of cardiogenic shock
induced by anti-asthma medication has no proven benefits if patients
maintain their mobility. Older asthma patients with cardiovascularcomorbidities and with well-controlled asthma are less likely to experience exacerbations and have a good quality of life.

REFERENCES

1. GAMA.Updated guide for asthma management, SRP guide 2007.
2. Marianne F. Distinguishing Asthma from a Heart Condition. FAAAAI.
June 2007.
3. European Society of Cardiology, ESC, Guide, J Eur Preventive Cardiology and Cardiac Rehabilitation 2007;14(2):1-40.
4. NAEPP Working Group, Bethesda, National Heart, Lung and Blood
Institute. Considerations for Diagnosing and Managing Asthma in the
Elderly 1996;1:5-12.
5. Cruickshank JM. Beta blockers continue to surprise. Eur Heart J
2000; 21:354-64.
6. Waldo A et al. Effect of d-sotalol on mortality in patients with left
ventricular dysfunction after recent and remote myocardial infaction,
Lancet 1996;348;7-12.
7. Brookes L, SENIORS, Study of the Effects of Nebivolol Intervention on Outcomes and Rehospitalisation in Seniors With Heart Failure,
Medscape Update 2004.
8. Salpeter SR, Ormiston M, Salpeter EE. Cardioselective β-Blockers
in Patients with Reactive Airway
Disease A Meta-Analysis. Ann
Intern Med , 2002;137:715-725.
9. Frishman W.et al. Comparison of controlled on sent extended release verapanil with amlodipine and amlodipine plus atenolol on exercise performance and ambulatory ischaemia in patients with stabile
angina. Amer J Cardiol, 1999; 83:507-14.
10. Braunwald E. Heart Disease: A textbook of Cardiovascular Medicine, Philadelphia,
1997;1196-98.
11. Nadrous HF, Ryu JH. Impact of Angiotensin-Converting Enzyme
Inhibitors and Statins on Survival in Idiopathic Pulmonary Fibrosis.
Chest 2004;126: 2 438-446.
12. Dykewicz MS. Cough and Angioedema from ACE Inhibitors: Coughand Respiratory Reactions. Curr Opin Allergy Clin Immunol 2004; 4.
13. Frieri M. Distinguishing Asthma From a Heart Condition. FAAAAI,
June 2007.
14. Ohnishi A, Kato M, Kojima J. Differential Pharmacokinetics ofTheophylline in Elderly Patients. Drugs&Aging 2003;20:71-84.
15. Weinberger M, Handles L. Theophylline in Asthma. The New Engl.
Journal Med 1996; 334(21):1380-88.
16. Roberts J, Snyder DL, Friedman E. Pharmacology of aging, A textbook, Sec Edition 1996; 111-17.
17. Global Initiative for Asthma Guidelines, GINA 2002.
18. Barnes P. Theophylline-New Perspectives for an old Drug, Asthma
In General practice. Am J Resp Crit Care Med 2003; 167: 813-818.
Salpeter SR, Ormiston TM, Salpeter EE, Poole PJ, cates CJ. Cardioselective Beta-Blocker for chronic obstructive pulmonary disease.
Respir. Med. 2003; 97(10): 1094-101.

IMPLICAŢIILE PE TERMEN LUNG ALE MEDICAŢIEI
CARDIOVASCULARE ŞI RESPIRATORII LA VÂRSTNICII ASTMATICIREZUMAT

Astmul bronşic la vârstnici, dar şi celelalte grupe de vârstă, prezinta o incidenţă crescută în ultimii ani. Comorbidităţile frecvente,în special în patologia cardiovasculaă, la vârstnici impun un tratament combinat, de multe ori cu reacţii adverse sau contraindicaţii
pentru celelalte boli asociate. De cele mai multe ori, se renunţă la medicaţia astmului ori la un medicament important din terapia
cardiovasculară din precauţie sau din considerente nejustificate, determinate şi de o politică prea exhaustivă şi uneori eronată
a vânzării medicamentelor la noi în ţară. Scopul lucrării este de sensibiliza şi delimita aceste aspecte, de a demonstra că este
necesară medicaţia fiecărei afecţiuni în parte, ţinând cont de reacţiile adverse sau contraindicaţiile respective, fără a omite tratarea
astmului la vârstnici, ţinta principală la această categorie de pacienţi, fiind menţinerea unei calităţii bune vieţii.
Cuvinte cheie: medicatie cardiovasculara, astm, varstnici

30

Fiziologia - Physiology  2010.20.2 (66)STROMAL CELLS - TUMOR MICROENVIRONMENT
INTERACTIONS - Part IIFLORINA BOJIN1, CALIN TATU2,3, OANA GAVRILIUC1, ALEXANDRA GRUIA3, GABRIELATANASIE1,3, CARMEN TATU1,3, ANGHEL SIMONA1,3, MIRABELA CRISTEA3, FELICIACIUCULESCU1, ALEXANDRU TOCUT1, DANIELA CRISNIC1,3, ADRIAN CARABINEANU4,DACIANA NISTOR1,3, CARMEN BUNU1, VIRGIL PAUNESCU1
1
Physiology-Immunology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Biology Department “Victor Babes” University of Medicine and Pharmacy, Timisoara
3
Regional Center for Transplant Immunology, Emergency Clinical County Hospital Timisoara
4
Oncologic Surgery Department, Municipal Emergency Hospital Timisoara

ABSTRACTThe complex cellular tumor (micro)environment comprises immunocompetent and inflammatory cells, endothelial cells and fibroblasts. All of these cell types may critically influence the multi-step process of carcinogenesis and malignant phenotype. Fibroblasts
are known to take part in immune reaction during tissue damage and injury by modulating local cellular and cytokine milieu, adjusting
the kinetics and components of the inflammatory infiltrate, and by modulating the functional status of the immunocompetent cells.
Purpose of our study was investigation on tumor-associated fibroblasts (TAFs) role in immune suppression, thus profoundly affecting
tumor cells progression.
Human TAFs were isolated from breast cancer surgical pieces, cultivated and expanded in vitro as monolayer cell culture. Supernatants of cell cultures at different passages were collected and presence of IL-4, IL-10, IL-13, TGF-β1, TNF-α, INF-γ and VEGF was
detected by ELISA method. Specific mouse anti-human fluorochrome-conjugated antibodies identified in flow-cytometric analysis
surface markers of TAFs, such as CD14, CD117, CD90, CD106, CD44, CD29, CD73, HLA-DR, CXCR4. Expression of cytoskeleton
and extracellular matrix proteins was revealed by immunocytochemistry (vimentin, α-smooth muscle actin).
Secretion of cytokines with direct immunosuppressive effect, such as IL-10 and IL-13, and other cytokines, IL-4 and TNF-α, was increased in cultured TAFs at all passages. Enhanced production of TGF-β1 by TAFs may also be a critical factor in tumor homeostasis.
Expression of MHC class II molecules (HLA-DR) on activated TAFs contribute to an additional level of immunosuppression and pro-tumoral effect. High level of VEGF production suggests that TAFs provide not only structural support, but also pro-angiogenetic molecules
for tumor vascularization. α-smooth muscle actin expression in fibroblasts is variable and associated with an activated status.
The complex network of effects TAF transduced by enhanced expression of various immune cell deactivating/suppressing factors
and neoangiogenesis molecules should stimulate consideration of TAFs-based therapeutic designs.

Key words: MSCs, TAFs, microenvironment
INTRODUCTION
Cancers may arise from malfunctions in the cell control
segment, that lead to the dysregulation proliferation of cells. The
underlying causes of cancers are mutations and other alterations in DNA, and attendant innappropriate expression level, ofgenes encoding proteins, that either promote growth or restrain
it, or direct the apoptosis machinery, or are responsible for DNA
damage repair and signaling, and chromatin remodeling. The
mutations can be inherited (present in the germ cells), or they
may be produced in somatic cells (1).
In metastasis, malignant cancer cells break away from
where they are immobilized, enter the circulatory system, andinvade other organs. They form colonies, or secondary tumors,at the new locations and cause damage to their neighbors byappropriating sources of nutrition (1).
Cancers derived from epithelial cells are the most common
type of cancer. In order for the cancerous epithelial cells to
break away from the initial location, they must detach from otherepithelial cells and from the ECM (2). To do so they must disrupt
the cell-to-cell adhesive contact established by E-cadherins, andcell-to-ECM contacts maintained by the integrins. Once they
have detached from one another and from the ECM they haceto pass through the basement membrane to reach and enter
the circulatory system.
Growth factors ad growth factor receptors support the development of malignant tumors in several ways. One proeminentcontributor to the onset of malignancy is the vascular endothelial
growth factor (VEGF). In order for a solid tumor to grow andthrive it must have an adequate blood supply. In response tothis need for vascular expansion, tumor angiogenesis take place.
The expression of RNA for VEGF ligands is enhanced in mosthuman tumor cells. Increased VEGF mRNA are present in rapidly
growing glioblastoma multiform brain tumors, and in cancersof the lung, breast, gastrointestinal tract, female reproductive
organs, thyroid gland, and urinary tract (3).
Alterations in the mix of cell adhesion molecules being ex-

Received January 2010. Accepted February 2010. Address for correspondence: Florina Bojin MD, Physiology-Immunology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, 2A
Eftimie Murgu Square, RO-300041, Phone/Fax: +40256490507, e-mail: florinabojin@umft.ro2010.20.2(66)  Fiziologia - Physiology

31pressed accompany tumor progression. The altered expression
Total RNA was extracted from MSCs and TAFs, at different
patterns occur not only during metastasis, but also during solid
passages, as well as from cells in coculture, at the end of 2
tumor growth. Most of the cancer develop from epithelial cells,
weeks period, from all cocultures types. From cultured cells, afterand loss of E-cadherins is a common occurrence. A consequence
trypsinization and washing with saline solution (PBS) followed byof the growth factor receptor-adhesion molecule clustering is thecentrifugation, total RNA was extracted using GenElute™ Mamstrengthening of signals that would otherwise be too weak to elicit
malian Total RNA Miniprep Kit (Sigma). Concentration of eacha cellular response if conveyed by one or other alone (4-6).
sample was measured using Nanodrop ND-1000 spectrophotomOnce the tumor cells are reaching the appropriate milieu, ineter (Wilmington, DE, USA). For RT-PCR we used concentrations
different organs and tissues, depending on the homing abilitiesof 100 ng RNA/reaction and we amplified fragments of 554 bpof the cells, an interaction between stromal compartment and(CXCR4), 584 bp (VEGF), 220 bp (IL-4), 424 bp (IL-10), 315 bp
tumor cells is established, which could activate the entire arsenal(TGF-β1) and 486 bp (Thy1). The following program was usedof changes in tumor cells and stromal cells behavior, starting
for amplification: 50oC - 31’, 95oC - 15’, (94oC - 1’, 55oC - 1’,with adhesion molecules connections, up to dysregulation of
72oC - 1’) x 35 cycles, 72oC - 10’; samples can be thus stored
the entire geneprofile, for both weretumor cells,
and stromal cells
at 4oC for indefiniteThermal
time period. For
gene amplificationusedsequencesvalidetedin GENEBANK.
cycleswere weadjustedfor eacompartments (7).
primer pairs whose sequences were valideted in GENEBANK.
presents
theweresequencesof primers
used for
while
Table
sequencesvalidetedin GENEBANK.
Thermalcycleswere
for eaWe investigatedmodificationinducedduring stromal
cells
Thermalcyclesamplification,were
adjusted for
eachprimerpair.adjusted
TableIII enumeratinteraction withby
tumorRT-PCR.
cells onthe
phenotypicaland analysis,genesequencesof primers used
for amplification,
Forbehavior
dataofthetheproducts
arewhile
presents
sequencesprimers presents
usedforamplification
amplification,
while
Table
IIsubmitted
enumerat
expression pattern, in coculture models developed in vitro
Table II enumerates the samples analyzed by RT-PCR. For data
electrophoresis,
visualizedwith analysis,
Fluor-S™
MultiImager(BIO-RAD)UV systby RT-PCR. For
data analysis,
thethe amplification
products
are submitted
conditions.
amplification products are submitted to 2 % agarose
GelDocsoftware,visualized
which allows
ofthe results.
electrophoresis,
witha semi-quantitative
Fluor-S™
(BIO-RAD)gelelectrophoresis,MultiImager
visualizedanalysis
with Fluor-S™
MultiImager(BIO- UV syste
MATERIALGelDoc
AND METHODS
RAD)
UVsystem,andanalyzed
with
GelDocsoftware,which
software, which allows a semi-quantitative
analysis of the results.
1. Isolation and culture of MSCs, TAFs and bulk tumor
allows a semi-quantitative analysis of the results.
Tabelul I. Genes and primers sequences used for RNA amplification and analysis u
cells
TabelulI. Genes and primers
used forRNA amplification andand analysis u
All cellular types were isolated,cultured andfurther exTabelul
I. Genes
and primers
sequencesusedsequencesfor RNA
amplification
analysis using
RT-PCRpairs
Gene
Primerpanded as previously described by Paunescu V et al. (8) andCXCR4
F: 3’-CAGCAGGTAGCAAAGTGACG-5’
Bojin F et al. (9).
GenePrimer pairs
R: 3’-AGACGCCAACATAGACCACC-5’
CXCR4
F: 3’-CAGCAGGTAGCAAAGTGACG-5’
VEGF
F:
2. Immunophenotyping
R: 3’-CTACCTCCACCATGCCAAGT-5’3’-AGACGCCAACATAGACCACC-5’R:
3’-TGGTGAGAGATCTGGTTCCC-5’
MSCs and TAFs in coculture with bulk tumor cells wereVEGF
F: 3’-CTACCTCCACCATGCCAAGT-5’
IL-4
F:
trypsinized (Trypsin-EDTA, Sigma) at the end of the experiR: 3’-TGCCTCCAAGAACACAACTG-5’
3’-TGGTGAGAGATCTGGTTCCC-5’R:
3’-ACTCTGGTTGGCTTCCTTCA-5’
ment, at 14 days, centrifuged 7 min at 1500 rpm, and washedIL-4
F: 3’-TGCCTCCAAGAACACAACTG-5’
IL-10
F:
twice with PBS. Cells were then re-suspended in PBS, 100 μl of
R: 3’-TTACCTGGAGGAGGTGATGC-5’
3’-ACTCTGGTTGGCTTCCTTCA-5’
R:
3’-TGGGGGTTGAGGTATCAGAG-5’
cellular suspension togheter with 4 μl of each antibody (mouse
IL-10
F: 3’-TTACCTGGAGGAGGTGATGC-5’
Thy1
F:3’-ATGAACCTGGCCATCAGCATCGC-5’
anti-human fluorochrom conjugated). Cells are then vortexed
R: 3’-TGGGGGTTGAGGTATCAGAG-5’
R:3’-TCACAGGGACATGAAATCCGTGG-5’
Thy1
F:3’-ATGAACCTGGCCATCAGCATCGC-5’
and put to rest at room temperature, in the dark, for 30 minutes.
TGF-β1F:
3’-GACTGCGGATCTCTGTGTCA-5’
R:3’-TCACAGGGACATGAAATCCGTGG-5’
After incubation, cells are washed with 2 ml Cell-Wash Solution
R:
3’-CCTCCCTTAACCTCTCTGGG-5’
TGF-β1
F: 3’-GACTGCGGATCTCTGTGTCA-5’(BD Biosciences), centrifuged 1 min at 1500 rpm, supernatant is
R: 3’-CCTCCCTTAACCTCTCTGGG-5’
removed and cells are re-suspended in 500 μl Cell-Wash. Flowcytometric analysis was performed using FACSCalibur (BectonDickinson), 2 lasers acquisition system, capable to differentiate 4
Tabelul II.Tabelul
Samplesanalyzed
forexpression
gene expression
II. Samples
analyzed for genecolors simultaneously. The antibodies used for flowcytometric asTabelul II. Samples analyzed for gene expression
say were conjugated with the following fluorochroms: PE - CD14
No..
Sample typeRNA conc.
(BD Pharmingen™), CD117 (BD Pharmingen™), α-SMA (BD Bio(ng/μl)
No..
Sample type
RNA
conc.
sciences), CD29, CXCR4, Nestin, E-Cadherin (R&D Systems),
1.
TAF cocultures-TAF medium
269.4(ng/μl)and FITC - CD34, CD44, CD45, CD73, CD90, CD106, HLA-DR,
2.
TAF
medium50.7
1.
TAF cocultures-tumor
cocultures-TAF medium
269.4(BD Pharmingen™), Cytokeratin (R&D Systems) şi APC - CD31
3.
TAF
1656
2.
TAF P5
cocultures-tumor medium
50.7(BD Pharmingen™). Part of the cellular markers (E-Cadherin,
4.
MSC
cocultures-MSC medium
204.3
3.
TAF P51656
Cytokeratin) have an intracellular location, recquiring a supple5.
MSC
medium58.4
4.
MSC cocultures-tumor
cocultures-MSC medium
204.3
mentary permeabilization step, using FACS Permeabilizing
6.
MSC P4574.7
5.
cocultures-tumor medium
58.4
Solution 2 (10x; BD Biosciences), followig a protocol presented
6.
MSC P4574.7
by the supplier. Acquisition and data analysis were performed
using CellQuest Pro software (Becton-Dickinson).
4. Statistic analysis
Statistic analysis was performed using Excell Microsoft
3. Characteristic gene expression of MSCs and TAFs in
Office2003 (Microsoft Corporation) software. The central
4.
Statistic
analysiscoculture system
32

4. Statistic
Statisticanalysis
analysis was performed using Excell Microsoft Office 2003 (Fiziologia- Physiology 2010.20.2(66)
Statistic
was performed
using
ExcellMicrosoft
Office2003
software. The analysis
central tendenciesof the variables
wereexpressedasa mean(M),(
software.
central (sd).
tendencies
of totheperform
variables
expressed
as a mean(M),
asstandardThedeviation
In orderthewerestatisticcomparisons,„t”-Studas standard
deviationwere(sd).used
In orderto perform variables.
the statisticDifferencescomparisons,„t”-Stude
analysis(ANOVA)for continuous
were
considanalysis(ANOVA)
were
used
for
continuous
variables.
Differenceswereconsid0.05.

tendencies of the variables were expressed as a mean (M),and the dispersion ones as standard deviation (sd). In orderto perform the statistic comparisons, „t”-Student test and thevariance analysis (ANOVA) were used for continuous variables.
Differences were considered significant for p < 0.05.
RESULTS
1. Characteristic gene expression of MSCs and TAFs in
coculture model with tumor cells
Gene expression was evaluated for both the cellular substrate MSCs and TAFs, as well as for tumor cells put in coculture
with them. The samples’ order is presented in Table II, from left
to right.

Fig.1. VEGF expression (left) and CXCR4 (right). Notice the increased
expression of VEGF in cocultures having TAFs as cellular substrate, while
MSCs present a decreased expression in coculture, independently of the culture media. MSCs cultured in appropriate medium do not present expressionof this molecular marker. CXCR4 is present in all cellular types.

VEGF expression is increased in TAFs (also confirmed byimmunocytochemical analysis); presence of this pro-angiogenic
factor in coculture model could be suggestive for TAFS’ role insupporting tumor cells. MSCs do not posses the same molecularcharacteristics as TAF, VEGF not being expressed in these cells;but, MSCs cocultures with tumor cells do present a comparable
expression of this marker, probably do to presence of tumor cells
characteristics (Figure 1).
CXCR4 is present in all cellular types, both independently
cultured or in cocultures. CXCR4 is a cellular homing marker,
MSCs and TAFs expressiong this receptor type on the cellularsurface.
IL-4 gene expression is very week, the results being correlated with the other analysis and tests performed for determinationof this cytokine (ELISA) (Figure 2). IL-10 is a molecular marker
present for all samples, while TGF-β1 is especially expressed in
TAFs and cocultures using this cellular substrate, MSCs laking
expression of this marker (Figure 3).

Fig.2. IL-4 expression. Decreased expression of IL-4 for MSCs at P4

Fig.3. IL-10 (left) and TGF-β1 (right) expression. Increased expression of IL10 for all samples, but decreased / absent for TGF-β1, in MSCs in coculture
or single culture, in presence of specific medium

Thy1, also known as CD90, is expressed in all samples. The
resultant protein is CD90 cell surface marker, which is present
on the surface of mesenchymal stem cells and tumor-associatedfibroblasts. Expression of this marker even in coculture is significant for MSCs and TAFs ability to retain their initial characteristicfeatures (Figure 4).

Fig.4. Increased and constant Thy1 (CD90) expression in all samples, significant for cellular ability to retain initial characteristics

2010.20.2(66)  Fiziologia - Physiology

33

3.4. Phenotypical analysis of cells in coculture
We used specific markers of MSCs and TAFs for phenotypical analysis of cells in coculture; identification markers of tumor
cells were also used. We could not find any significant differences in markers expression in MSCs coculture systems, fo both
medium types used – MSCs specific medium and tumor cells
medium (Table III, Figures 5 and 6). TAFs cocultures with tumor
cells presented significant differences in markers expression
when cultivated in fibroblasts specific medium, compared withtumor cells medium. Cytokeratin, CXCR4, HLA-DR and α-SMA
expressions were decreased, while CD44 and CD90 expression
presented a significand increased compared with TAFs cultures
alone. When we compared the two cellular types in coculture withokeratin, CXCR4,
HLA-DRand α-SMA
expressions
werecomplementary
media, significant
differenceswere revealed
in decreased, while CD44 andpresented aCD45
significand
increasedstemcomparedcultures alone. When we
expression (hematopoietic
cells marker),withmeningTAFs
that
the coculturesusing MSCs
as cellular
substrate presented
an significant differences were
cellular types
in coculture
withcomplementary
media,
overexpression of CD45,
for bothmediumtypes, whencompared
expression (hematopoietic
stem
cells
marker),meningthat the cocultures using MSCs
withcorrespondent
cocultures
using
TAFs
as
cellularsubstrate.
te presented an overexpression of CD45, for both medium types, when compared withCXCR4, which was detected as marker for TAFs coculture
ultures using
TAFs
as cellular
substrate.
CXCR4,
systems,presented
a lower expression
comparative
withwhich
MSCs was detected as marker forystems, presented
lower when
expression
comparativewith MSCs cocultures, especiallycocultures,aespecially
we used tumor
cells specific medium
or cells specificmediumfor(Table III, Figures 7 and 8).
for cultivation(TableIII, cultivation
Figures 7 and 8).

Fig.6. Phenotypical surface markers of MSCs-tumor cells cocultures in tumor cellFig.6.markers
Phenotypicalmarkers of MSCs-tumor
cells coculturesinFig.6. Phenotypical surface
ofsurfaceMSCs-tumor
cells coculturesin tumorcells growt
tumor cells growth medium

Phenotypical surface
markersof MSCs-tumor
cellscells
cocultures
MSCs’ growth medium
Fig.5. Phenotypicalsurfacemarkers of MSCs-tumor
cocultures ininMSCs’
growth medium

34

Fiziologia - Physiology  2010.20.2 (66)Fig.8. Phenotypical surface markers of TAFs-tumor cells cocultures in tumor cells’ growTabel III. Synopsis of flowcytometric markers in MSCs/TAFs – tumor cells

cocultures
Tabel III. Synopsis of flowcytometric markers
in MSCs/TAFs – tumor cells cocu

Medium type

MSCs
medium
(%)

CD14
CD29
CD44
CD45
CD90
CXCR4
HLA-DR
Citokeratina
Nestina
α-SMA
Cellular type

33.90
50.24
68.66
32.91
94.44
31.84
MSCs

Tumor
cells’
medium
(%)
34.22
70
66.04
33.52
86.88
31.29
MSCs

TAFs
Medium
(%)
37.95
1.42
69.18
41.37
44
34.47
18.87
33.95
TAFs

Tumor
cells’
medium
(%)
65.45
1.33
77.49
14.95
26.52
21.29
18.79
23.94
TAFs

DISCUSSION AND CONCLUSIONS
The in vitro model of cocultures between MSC / TAF and bulk
Fig.7. Phenotypical surface markers of TAFs-tumor cells cocultures in TAFs’
DISCUSSIONANDCONCLUSIONS
tumor cells
proves medium
to be a functional model for analysis of tumor
ypical surface markers of TAFs-tumor
coculturesin TAFs’
growth
growth medium cells
cells behavior, similar to the conditions encountered in vivo. MSCs
and TAFs, used as cellular substrate in the coculture models exhibit
The in vitro model of cocultures
between MSC / TAF and bulk tumor cells pro
a profound alteration of morphological aspect, induced by culture
model for analysis of tumor cells
behavior,
similarthe conditions
conditions.
Morphologicalchanges toare associatedwith alterationsencounteredphenotypicalmarkers expressionof cellsexhibit
in coculture,a asprofoundwell asTAFs, used as cellular substrateof in
the coculture
modelsalteratmodifications
insectretor
profileofthesecells.
MSCs
retaintheinitialaspect, induced by culture conditions. Morphological changes are associatedcharacteristic of stem cells (CD90, CD44 expression), while TAFs
phenotypical markers expressionpresentof cells
in coculture,as markers
well asin seca decresed
expression of thesewhenmodifications
cultured withcells. MSCs retain the initial characteristic
of
stem
cells
(CD90,
CD44
expression),tumor cells, in coculture conditions. Several cytokines are secreted
within the culture
medium,but the results
converge
to the conclusion
decresed expression of these markers
whenculturedwithtumor
cells, in coculturthat they caould be responsible for decreased cellular proliferation,cytokines are secreted within theculture medium, but the results converge to thefor both cellular substrate components, as well as for tumor cells.
caould be responsible for decreased
for toboth
cellular
Thecellular
hypotesis of proliferation,
tumor-fibroblast interactions
criticallydepend substrateon fibroblast and tumor cell type, as well as on the proportion of
as for tumor cells.
coculturedinteractions
cells was elaborated
by Olumi et al.depend
since 1998on(10).fibroblast a
The hypotesis of tumor-fibroblast
to critically
A tumor-promoting effect of TAFs is supported by the more recent
well as on the proportion of cocultured
was elaborated
by whoOlumi
in vivo and in cells
vitro observationof Spaeth et al. (11),demon-et al. since
strated tumor-derived fibroblasts to suppress tumor cell apoptosis.
Although being inconsistent, the literature data clearly imply thesource of fibroblasts (normal, embryonic, tumor-derived) as a critical determinant in tumor cell response. Both cell-cell and cell-matrix
interactions, as well as paracrine factors are found to be involved inthis dynamic process.
According to the expression profile depicted in our previous
paper, TAFs produce a multitude of paracrine immune-modulators,
including chemokines, cytokines and peptide growth factors (8,9),
while the correspondent gene expression seem to be increased foralmost all these factors, while TAF/MSC are put in contact with tumor
cells. Lack of deactivation of fibroblasts contributes to persistenceof inflammatory infiltrate and prolonged inflammation. Accordingly,
stromal cells are considered santinels in chronic inflammation as theyessentially contribute to leukocyte migration to the site of damage
and influence the local immune response (12,13). It is well known
that chronic inflammation and malignancies show some molecularand cellular similarities with the regard to cytokine profile (14,15).
MSCs and TAFs could induce an increase of the in vitro proliferationof tumor cells, but their effect could be different in vivo, due
Fig.8. Phenotypical surface markers of TAFs-tumor cells cocultures in tumor
tointerventionof the immune system. They profoundly contribute
cells’ growth medium

ical surface markers of TAFs-tumor cells cocultures in tumor cells’ growth medium
2010.20.2(66)  Fiziologia - Physiology

Synopsis of flowcytometric markers in MSCs/TAFs – tumor cells cocultures
cal surfaceMedium
markersof TAFs-tumor
cells coculturesin tumor cells’ growth medium
type
MSCs
Tumor
TAFs
Tumor
mediumcells’

Medium

MSCs

Tumor
(%)

TAFs

cells’

Synopsis of flowcytometric– tumor cells cocultures(%) markers
mediumin MSCs/TAFs(%)
mediumMedium type

Tumor
(%)

35

to the creation of a tumor microenvironment that inappropriately
promotes expansion and dissemination of neoplastic cell population
by determining a tumor-specific profile.
Far from being complete, our present study gives an overview
and discusses how MSCs and TAFs directly and indirectly affecttumor growth and propagation in order to stimulate consideration ofstromal cells as novel therapeutic target in solid tumors. Recapitulation the modified expression profile of MSCs and TAFs in coculture
with normal corresponding cells, some examples are given to stressthe potential impact of stromal cells, especially TAFs on tumor
progression, i.e. TAF-derived signals resulting in angiogenesis andimmune dysfunction.
Acknowledgements
This work was supported by PNCDI-II – Parteneriate, project
number 41-019/2007 and by CNCSIS –UEFISCSU, project number
PNII – IDEI 1748/2008
REFERENCES

1. Haddow A. Molecular repair, wound healing and carcinogenesis: tumor
production a possible overhealing? Adv Cancer Res 1972; 16: 181-234.
2. Birchmeier C et al. MET, metastasis, motility and more. Nature Rev. Mol.
Cell Biol. 2003; 4: 915-25.
3. Comoglio PM, Boccaccio C, Trusolino L. Interactions between growth
factor receptors and adhesion molecules: Breaking the rules. Curr. Opin.
Cell Biol. 2003; 15: 565-71.
4. Canacci-Sorrel M, Zhurinsky J, Ben-Ze’ev A. The cadherin-catenin adhesion system in signaling and cancer. J. Clin. Invest. 2002; 109: 987-91.

5. Nagase H, Woessner JF, Jr. Matrix metalloproteinase. J. Biol. Chem.
1999; 274: 21491-94.
6. Stamenkovic I. Extracellular matrix remodeling: The role of matrix metalloproteinase. J. Pathol. 2003; 200: 448-64.
7. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, et al. Bonemarrow-derived mesenchymal stem cells as vehicles fro interferon-betadelivery into tumors. Cancer Res 2002; 62: 3603-3608.
8. Paunescu V, Bojin MF, Tatu CA, Gavriliuc O, Rosca A, Griua A, Tanasie
G, Bunu C, Crisnic D, Gerghiceanu M, Tatu CS, Tatu RF, Vermesan S.
Tumour-associated fibroblasts and mesenchymal stem cells: more similarities and differences. JCMM 2010
9. Bojin F, Tatu CA, Gavriliuc O, Gruia A, Anghel S, Tanasie G, Tatu C, Cristea M, Ciuculescu F, Tocut A, Crisnic D, Carabineanu A, Nistor D, Bunu C,
Paunescu V. Stromal cells-tumor microenvironment interactions – Part I.
Fiziologia-Physiology 2010; 20 (65): 37-41.
10. Olumi AF, Dazin P, Tisty TD. A novel coclture technique demonstrates
that normal human prostatic fibroblasts contribute to tumor formation of
LNCaP cells by retarding cell death. Cancer Res. 1998; 58: 4525-30.
11. Spaeth EL, Dembinski JL, Sasser AK, Watson K, Klopp A, Hall B, Andreeff M, Marini F. Mesenchymal stem cell transition to tumor-associatedfibroblasts contributes to fibrovascular network expansion and tumor progression. PLoS One. 2009; 4(4): e4992.
12. Murakami OS, Okada H. Lymphocyte-fibroblast interactions. Crit. Rev.
Oral Biol. Med. 1997; 8: 40-50.
13. Smith ORS, Smith TJ, Phipps RP. Fibroblasts as sentinel cells. Synthesis of chemokines and regulation of inflammation. Am. J. Pathol. 1997;
151: 317-322.
14. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow?
Lancet 2001; 357: 539-45.
Kunz-Schughart LA, Knuechel R. Tumor-associated fibroblasts: functional
impact on tumor tissue. Histol. Histopathol. 2002; 17: 623-37.

INTERACTIUNEA DINTRE MICROMEDIUL TUMORAL SI
COMPARTIMENTUL STROMAL – Partea II
REZUMAT
Micro-mediul celular tumoral complex include celule imunocompetente si inflamatorii, celule endoteliale si fibroblaste. Aceste tipuri
celulare ar putea avea o influenta critica in procesul stadial al carcinogenezei si inducerea fenotipului malign. Se stie ca fibroblastele
participa la reactiile imune din timpul injuriei tisulare prin modularea mediului local celular si citokinic, ajustand cinetica si componentele infiltratului inflamator, precum si prin modularea statusului functional al celulelor imunocompetente. Scopul acestui studiu a fost
investigarea rolului fibroblastelor tumorale (TAF) in imunosupresie, cu efect important asupra dezvoltarii celulelor tumorale.
TAF umane au fost izolate din piese de rezectie chirurgicala provenite din cancere mamare, fiind cultivate si expandate in vitro in culturi
celulare monostrat. Supernatantele culturilor celulare la diferite pasaje au fost colectate si determinata prezenta IL-4, IL-10, IL-13,
TGF-����������������������������������������������������������������������������������������������������������������������������������
β���������������������������������������������������������������������������������������������������������������������������������
1, TNF-��������������������������������������������������������������������������������������������������������������������������
α�������������������������������������������������������������������������������������������������������������������������
, INF-�������������������������������������������������������������������������������������������������������������������
γ������������������������������������������������������������������������������������������������������������������
si VEGF prin metoda ELISA. Pentru analize flowcitometrice au fost folositi anticorpi specifici conjugati cu fluorocromi, care sa identifice expresia markerilor de suprafata fibroblastici - CD14, CD117, CD90, CD106, CD44, CD29, CD73, HLA-DR,CXCR4. Expresia proteinelor citoscheletice si ale matricei extracelulare (vimentina, α-SMA) a fost detectata prin imunohistochimie.
Secretia citokinelor cu efect imunosupresor direct, IL-10 si IL-13, precum si a altor citokine, IL-4 si TNF-α, a fost crescuta pentru TAF
in cultura, indiferent de pasaj. Cresterea producerii de TGF-β1 in supernatantul TAF ar putea fi un factor important in homeostazia
tumorala. Expresia moleculelor CMH clasa II (HLA-DR) pe suprafata fibroblastelor activate contribuie suplimentar la imunosupresie
avand un efect pro-tumoral. Nivelul crescut de VEGF sugereaza ca fibroblastele tumorale nu confera doar support, ci si molecule
pro-angiogenice necesare neovascularizatiei tumorale. Expresia ���������������������������������������������������������������������
α��������������������������������������������������������������������-SMA este variabila si este asociata cu un status fibroblastic activat si agresiv. Complexa retea de efecte induse de TAF prin cresterea expresiei unor factori care dezactiveaza sau suprima celulele
imune ar putea fi considerata pentru strategii terapeutice viitoare bazate pe TAF.

Cuvinte cheie: MSC, TAF, micromediu
36

Fiziologia - Physiology  2010.20.2 (66)THE ANALYSIS OF STATE OF TENSION AND STRAIN AT THE
LEVEL OF CERVICAL VERTEBRAE (ATLAS) THROUGHFINITE ELEMENT METHODMACIOVAN ADINA
Family doctor, Soc.Civ.Med. DUO-MED, Timisoara, Romania

ABSTRACT
Objective: the analysis of state of tension and strain at the level of cervical vertebrae (atlas) through finite element method.
Method and material: We started with CAD modelling of biomechanic structures, realising a model of sustainability of the cervical C1
vertebrae, after which we performed the analysis states tension and strain at the level of cervical C1 vertebrae using finite element
method. For elaboration of the solid CAD model was used tomography scanning of a real human atlas. The model developed was
subjected to different loads through successive iterations similar to those governing the spine in a car accident.
Results: Higher or lower incidence of certain injuries is not random, and can not be charged solely to the conditions of the accident
occurrence, but is implicitly determined by the structural characters of the vertebrae and the size of the loading forces.
Key words: cervical vertebrae C1, tension, deformation, resistance

INTRODUCTION
The achievement of CAD models of the cervical vertebrae,
which covers the four basic phases of reverse-engineering in
the geometric modelling namely: data acquisition phase, stageof data pre-processing, the stage delimiting the areas andconstruction of the surfaces, construction phase of the solid
geometrical model CAD.
The phase of data acquisition treats the imagistic of thevertebrae transverse sections through the process of tomography scanning. The phase of data pre-processing sets in detailthe geometric reconstruction of the vertebrae surfaces startingfrom cross section images obtained by tomography scanning.
Reconstructed surfaces of the vertebrae form the triangulation
network, obtained by the technique of bevelling modelling, thistechnique being described in full in the introductory chapter.
MATERIALS AND METHODSIn this stage appear the itinerary realising the triangulation
network using the program of MATERIALISE Mimics 10.01.
It is important to mention that physical object surface detailfidelity, whose geometric reconstruction was performed after
its tomography scanning, depends on the accuracy of thecross sectional images of self-performance and implicit on
the performances of the tomography scanning equipment.
Tomography equipment of the last generation is capable ofvery high performance in terms of achieving cross-sectional
images of any physical object.
Therefore the use of performant tomography equipment inconjunction with the adoption of appropriate programs of geometric reconstruction leads to impressive results in the field. In

general, following completion of the triangulation network, there
may be defects of discontinuities (gaps) type, which necessarily
must be removed by filling operations. These operations were
performed under the Rapidform XOR program. After carryingout the operations of filling gaps and refining the triangulation
networks it results a number of 374,314 facets for the cervicalvertebrae, the average size of 0.2 mm sides. With in the phaseof areas delimitation and surfaces construction, triangulation
network is divided into a multitude of areas (regions) taking into
account the variation of the curves as there are oriented thefacets in space. Operation of delimitation of areas or regions is
also called segmentation. On the basis of regions resulting fromsegmentation operation is performed the3D curved surfacesthat mimic the geometry of the scanned object 3D, such areas
are interconnected by criteria of continuity and tangency. The
3D curved surfaces may be of Bézier or NURBS type. In otherwords, within the stage, it is realised the transition from themultitude of flat triangular surfaces (facets) of the triangulation
network to the multitude of 3D curved surfaces. In the case of
the C1 vertebrae, the stage of areas delimitation and creationof surfaces was conducted throughout the Rapidform XOR
program. Basically, were obtained 2.165 surfaces for the cervical vertebrae. In the future compatibility of Bézier surfaces with
CAD platforms, they are saved in an IGES file.
The CAD solid geometrical model construction phaserealises the compatibility at the level of representation andconsistency of Bézier surfaces with CAD platforms. This compatibility depends entirely on the type of the file that storesinformation about the Bézier surfaces, file made at the completion of the earlier phase. Therefore, generally the surfaces ofReceived May 2010. Accepted June 2010. Address for correspondence: Maciovan Adina, MD, PhD student, Family doctor, Eftimie Murgu Square No. 2A, 300041 Timisoara, Romania, Phone:+40256215584

2010.20.2(66)  Fiziologia - Physiology

37

Bézier type are saved in IGES files, these files having 100%After declaring the type of study and specification of thecompatibility with most CAD platforms. Also during this phasematerial characteristics, it was chosen the type of discretization
it is realised the ‘’hardening’’ of the geometrical model by taking
in the finite elements (operation called meshing). In this caseinto account the volume defined by the multitude of the Bézier
it were used finite elements of the tetrahedral type with four
surfaces and the possibility of linking this volume of some mass
nodes per element – TETRA 4 adopted for the FEM analysis ofproperties specific to a considered material. In case of thesolid bodies. It was adopted a high accuracy of discretization
cervical vertebrae, the operations afferent to the phase wereof the two CAD models in half of the vertebrae, the averagerealised in the CAD SolidWorks 2005 program. Analysis of thesize of finite elements was 1 mm. The discretization of thestress and strain states at the level of the cervical vertebrae
cervical vertebrae model gathered 100,141 finite elements with
with the help of the Finite Element Method (FEM) begins witha total of 19,479 nodes. Introducing the boundary conditions
a brief description of the method. The proximate character ofor conditions on the outline are materialized by declaring thethe FEM results pursuant to the fact that the real geometry is
bearings imposed on CAD models of the vertebrae (thosealways replaced with a network of finite elements seeking theareas considered fixed, that do not suffer of displacements
real form, but it can not be exactly reproduced only for someor who suffer certain blocked displacements - displacements
particular geometry, due to finite number of elements, andbeing targeted to the three fundamental axes: x, y and z). Also
the unknown measures of the problems are calculated only
during this phase, over the CAD models of the vertebrae arein structure nodes. It follows the conclusion that the accuracy
applied external charges (external forces – direction of the forceof calculation increases with the increase of the number ofmarked with the mauve shade on the figures) that take place
finite elements.
the appliance of some shocks ( strokes) as effect of somethe force marked with the mauveafter
shade
on the figures) that take place after the app
In general, structures of resistance, whose geometricsituations encountered in various road accidents.
( strokes)
as (i.e.
effectof someshape present a planeof symmetry
the structures
maysituations encountered in various road accidents.
be considered at geometric level as two ‘’halves’’ identical
RESULTS
mirrored),it is analysed with FEM at the level of a single ‘’half’’
A series of FEM analysis were made, each analysis taking
RESULTSof the considered structures. As to the conditions applied in theinto account a possible scenario as a result of a road accident.
FEMmodelanalysisIn were
made,
into
FEM analysis, one can say thatA
theseries
CAD solidof
geometric
each analysis,
the eachadoptedanalysis
value of the taking
external force
ap-account aon half of a symmetricalstructure
resistanceisembedded
in
plied
was
determinedbysuccessive
iterations,
so
that
the
total
result of a road accident. In each analysis, the adopted value of the external force a
the absent part (the half in the mirror) not represented in thevoltage σvonMises to reach the maximum allowable value (133by successive
so that thevoltageto reachthe maximum
model. For these reasons,
CAD models iterations,of the two vertebraeMPa),total
case inwhich it isσvonMises
considered the dangerof fracture
case
which
it isof theirconsidered
the dangerof fracture
occurrence– the red sha
were analyzed withMPa),
FEM taking
into inaccount
only half
occurrence– the red shadeon the figures
show the risk areas
geometry.
forfracture
occurrence.
The
possiblescenarios
considered
the risk areas for fracture occurrence. The possible scenarios consideredforfor the cervAs main objective of the analyses with FEM it consists thethe cervical vertebrae are:) frontal
bump(anterior)( Fig.1,
2, 3a,horizontallyand 3b),distribution method ofIthe
total tensions
throughout
each verte- horizontallyI ) frontal
bump (anterior)( Fig.1, 2, 3a, andbra in part, tensions due to forces developed in various impact
3b),II ) back bump on vertical plan ( Fig.4,
5),
situations on the vertebrae, based on the fact that maximumII ) back bump on vertical plan ( Fig.4, 5),values of tension during
known, onrespectively
com- plan (III Fig.6,) side bumpon horizontal
III )spreadsidearebump
horizontal
7, 8)
and plan ( Fig.6, 7, 8) andpression, supportedbybonematerial.
Also,
as
the
main
objecIV)
verticallyblow
aseffect of thecompressionvertebraeofIV ) vertically blow as effect of the lateral compressionof lateral
the cervical(F
tive is to determine the values of the critical forces of impact
the cervical vertebrae (Fig. 9, 10, 11).
that can cause fractures at the level of the C1 vertebrae.
The Software package with which the analyses with FEM
were made, within the CAD SolidWorks 2005 program, is entitled COSMOSWorks 2005. Initially it is specified the type ofstudy to be conducted. The type „Static Analysis’’ was chosenbecause it is considered that the forces that are developedon the vertebrae are applied in a static regime. Once thestudy defined, the material of which the assembly is made, isdeclared. Because the SolidWorks 2005 and COSMOSWorks
2005 package do not include in the database of the material
types the characteristics of the bone matter, a new sheet ofmaterial was created with the required characteristics (maximum tensions during stresses - compression, the longitudinal
modulus of elasticity and the coefficient of Poisson’s transverse
contraction). It is noted that it was considered perfectly homogeneous and isotropic material, i.e its mechanical propertiesof resistance do not vary with the directions of application
Fig.1.of Cervical vertebrae - frontal bump (anterior) horizontally – leaningFig.1. Cervical vertebrae - frontal bump (anterior) horizontally – leaning
forces. This fact was used as a simplifying assumption.
38

Fiziologia - Physiology  2010.20.2 (66)Fig.3. b) Cervical vertebrae - frontal bump (anterior) horizontally – deformations effects the red sha
Fig.3.b)b)Cervical
Cervicalvertebrae
vertebrae--frontal
frontalbump
bump (anterior)(anterior)horizontally–– deformations
risk areas
for fracture
occurrence effects
Fig.3.
horizontally
deformations
effectsthe
theredredsha
sh
risk
areas
for
fracture
occurrence
risk
areas
for
fracture
occurrence
Fig.1.
horizontally –– leaning
leaningFig.1. Cervical
Cervical vertebrae
vertebrae -- frontal
frontal bump
bump (anterior)(anterior) horizontally

Fig.4.–Cervicalvertebrae - back bump on vertical plan – leaningFig.2.
vertebrae-- frontal(anterior)horizontallycharges
Cervical vertebrae- frontalbump
bump (anterior)horizontally– charges Fig.4.
Fig.4.vertebrae
Cervical vertebrae- back bumpon verticalplan – leaningFig.2. Cervical
CervicalFig.2.
vertebrae
frontalbump(anterior)horizontally– Cervicalcharges- backbump
on verticalplan – leaning
Fig.4. Cervical vertebrae - back bump on vertical plan – leaningFig.5.
- backon vertical planee -- frontal(anterior)horizontally–(anterior)
deformations
effects thethe
red Cervicalshade
onvertebrae
the figures
figures
showbump
the onfrontal bump
bump
horizontally
deformations
effects
red
shade
on
the
show
the
Fig.5.
Cervicalvertebrae- backbump
vertical plan
Fig.3.(anterior)a) Cervical vertebrae- frontal bump –horizontally – deformaFig.5. Cervical vertebrae - back bump on vertical plan
risk
areas
for
fracture
occurrence
tions effects
the
red
shade
on
the
figures
show
the
risk
areas
for
fracturerisk areas for fracture occurrence
Fig.5. Cervical vertebrae - back bump on vertical plan
occurrence

Fig.6.
horizontal plan
plan –– leaning
leaningFig.6.Cervical
Cervical vertebrae
vertebrae -- side
side bump
bump onon horizontale - frontal bump(anterior) horizontally – deformations effects
red shadeon the figures
show thehorizontal plan – leaningFig.6.theCervicalvertebrae- sideFig.3. b) Cervical vertebrae - frontal bump (anterior) horizontally – deformaFig.6.
Cervical vertebrae- sidebump
bump on on
horizontal plan – leaning
risk
areas
fracture
occurrence
tions effects
the redshadeforon thefigures show
the risk areas for fracture
occurrence

2010.20.2(66)  Fiziologia - Physiology

39

Fig.9. Cervical vertebrae - lateral compression – leaningFig.7. Cervical vertebrae - side bump on horizontal plan
Cervical vertebrae������������������������������������������������������
�����������������������������������������������������
- side verticallybump on horizontal
plan vertically
blow asof
ef-the lateral co
Fig.7. Cervicalvertebrae- -side
side
bump
on horizontal
plan - sideplan
Fig.10.
Cervicalvertebrae
bumpFig.10.
on horizontal
plan
blow
as effect
Fig.7. Cervicalvertebraebump
on horizontal
fect of the lateral compression of the cervical vertebrae - deformations effects
vertebrae - deformations effects

Fig.11.
Cervical- sideplan
bumpFig.11.
on horizontal
plan
vertically blow as effect of the lateral cobumpvertebrae
on horizontal
Fig.8. Cervicalvertebrae- sideCervical vertebrae������������������������������������������������������
�����������������������������������������������������
- side bump on horizontal plan vertically blow as efFig.8. Cervical vertebrae - side bump on horizontal plan
Fig.8. Cervical vertebrae - side bump on horizontal plan fect of the lateralvertebrae- ofdeformations
compression
the cervical vertebraeeffects- deformations effects

CONCLUSIONS
Issue I): in case of an external bump applied to anterior sideof the atlas body, the fracture risk is highest to the posterior spring
CONCLUSIONS
and with maximum deformations to the anterior spring, the siteof action offorce (Fig.
2, 3a, andto
3b) anterior.
Issue I): in case of an external
bumpappliedside of the atlas bo
Issue II): in case of an external bump applied to the rearhighest to the posterior spring andsidewith
maximum deformations to the anterior spriof the atlas, to the posterior tubercle, the risk of fracture
force (Fig. 2, 3a, and 3b) .
occurrence is highest to the anterior spring and the maximum
deformations to the posterior spring, the site of action of force
Issue II): in case of an externalbump applied to the rear side of the atlas, to(Fig. 4, 5) . A similar force acts on the posterior spring direction
the risk of fracture occurrence ismovement
highestto the anterior
and thehyperextension
forced back byspring
the head, following
a maximu
forcedflexion,given
byfrontalimpact.
posterior spring, the site of action of force (Fig. 4, 5) . A similar force acts oIssue III): in case of an external lateral bump applied to thedirection movement hyperextension
forcedback thebyriskthe
head,occurrence
followingtransverses
apophyses,of fractureis high-a forced fl
impact.
est to the lateral pedicles and the maximum deformations at thecompression–
leaningFig.9. Cervicalvertebrae
lateraltransverses apophyses, in the site of action of force (Fig.6,7, andFig.9. Cervical vertebrae - lateral compression – leaning
III): in caseof an externallateral bump applied to the transverses a
Fig.9. Cervical vertebrae - Issue
lateral compression– leaning
8). Such a force can be printed by forced flexion movement of thefracture occurrence is highest tohead,theorlateralpediclesmaximum deformat
by direct kick,side impactand
printerthe
car accident.
Issue
IV):
in
case
of
an
external
bumpinverticalplan with can be pri
apophyses, in the site of action of force (Fig.6,7, and 8). Such a force
40

movement of the head, or by direct kick, sideFiziologia
impact- Physiology
printer car
accident.
 2010.20.2(66)
Issue IV): in case of an external bump in vertical plan with the lateral ccervical vertebrae to the transverses apophyses, the risk of fracture occurrence i
place and increase to the vertebral spring; the maximum deformations is to the tran

the lateral compression effect over cervical vertebrae to thetransverses apophyses, the risk of fracture occurrence is highest to the shock place and increase to the vertebral spring; themaximum deformations is to the transverse apophysis (Fig. 9,10, and 11). Such a mechanism is involved in producing Jefferson fracture type.
After conducting the analysis with MEF afferent to each scenario in hand, it can come off a first conclusion, perhaps the mostimportant one, the one related to the resistance of the vertebrae,the maximum resistance being given by the highest value of theexternal load applied in various situations of possible scenarios.
Thus, the cervical vertebrae presents the maximum resistancein case of a horizontal lateral bump (734 N) and the minimum
resistance in case of a vertically back bump (260 N).
In terms of deformations (movements) for maximum results,
we considered favourable the situations in which they recorded
the minimum values. Thus, the cervical vertebrae presents theminimum resultant deformation in the case of a vertical stroke
as effect of lateral compression of the cervical vertebrae (0.515mm) and the maximum resulting deformation for in the case ofvertically posterior bump (2.646 mm).
The results of this study demonstrating that higher or lower
incidence of certain injuries is not random, and can not becharged solely to the conditions of the accident occurrence,but is implicitly determined by the structural characters of thevertebrae and the size of the loading forces.
This study was included into the PhD thesis entitled”Clinico-statistical considerations regarding the incidence
and emergency treatment of fractures of the cervical andlumbar vertebrae caused by car accidents”, developed bydoctoral student under Maciovan Adina, scientific supervisor:
university teacher dr. Pompiliu Petrescu, U. M.F. “Victor Babes“ Timisoara, dec. 2009.
REFERENCES

1. Culea L. Concept of inverse engineering in modeling of biomechanicalstructures – PhD. Thesis, Timisoara, 2009.
2. Diaconescu N, Veleanu C, Klepp HJ. The spine – structure and function,
1977, Ed. Medicala, Bucharest.
3. Dragulescu D. Modeling in biomechanics, 2005, Ed. Didactica andPedagogica, Bucharest.
4. Ehara S, el-Khoury GY, Clark CR. Radiologic evaluation of dens fracture: Role of plain radiography and tomography. Spine  1992; 17:475479.
5. Prundeanu A, Vermesan H, Prundeanu H. Politrauma, 2nd Edition,
2001, Ed. Mirton, Timisoara.
6. Vermesan H. Traumatology of spine, pelvis and inferior limbs, 1997,
Ed.Mirton, Timisoara.
7. Vermesan H, Prundeanu A, Prundeanu H. Politrauma, 2nd Edition,
Vol.I, 2001, Ed. Mirton, Timisoara.

ANALIZA STARII DE TENSIUNE SI DEFORMATIE LA NIVELUL VERTEBREICERVICALE ATLAS CU AJUTORUL METODEI ELEMENTULUI FINITREZUMAT
Obiectiv: analiza starilor de tensiune si deformatie la nivelul vertebrelor cervicala C1 cu ajutorul metodei elementului finit.
Material si metoda: Am pornit de la modelarea CAD a structurilor biomecanice realizand un model experimental solid al vertebrei
cervicale C1, dupa care am efectuat analiza starilor de tensiune si deformatie la nivelul vertebrei cervicale C1 cu ajutorul metodei
elementului finit. Pentru elaborarea modelului solid CAD s-a apelat la scanarea tomografica a unui atlas real uman. Modelul realizata fost supus prin iteratii succesive la diferite incarcari, similare cu cele la care este supusa coloana vertebrala intr-un accident deautomobil.
Rezultate: incidenta mai mare sau mai mica a anumitor leziuni nu este intamplatoare, si nu poate fi pusa numai pe seama conditiilor de producere a accidentului, ci este implicit determinata de caracterele structurale ale vertebrelor si de marimea fortelor deincarcare.
Cuvinte cheie: vertebra cervicala C1, tensiune, deformatie, rezistenta

2010.20.2(66)  Fiziologia - Physiology

41

SEROLOGICAL IMMUNOPATHOLOGY OF PATIENTS WITH
MYASTHENIA GRAVISBOGDAN ISTRATE1,2, CRISANDA VÎLCIU2
1
Department of Neurobiology and Molecular Physiology,University of Bucharest, Romania
2
Department of Neurology,Clinical Institute Fundeni,University of Medicine and Pharmacy, Bucharest, Romania

ABSTRACT
Acetylcholine receptor (AChR) antibodies are present in sera from 80-90% of patients with generalized Myasthenia Gravis. Besides
AChR antibodies, in serological immunopathology of myasthenia, other antibodies are incriminated, such as MuSK, anti-titin, antiryanodine receptor (RyR), anti striational antibodies. These antibodies are responsible of complement activation. In mysthenic sera
complement components like C3 and C4 are detectable too in variable concentrations, depending by the medication and the clinicalcourse of the disease. MG is treated with cholinesterase inhibitors or/and with immunosuppressive drugs. Surgical removal of thethymus (thymectomy) may result in permanent remission or reduced needs of drug therapy. These factors mentioned above could
modify in a significant way the immunoserological profile of the myasthenic patients, allowing in the same time a good monitoringof the disease.
Key words: myasthenia gravis, serologic tests, immune markers

INTRODUCTION
Myasthenia gravis is a chronic autoimmune disease that
results in progressive fatigue, loss of muscular tonus andincreasing paralysis. These symptoms are caused by inappropriate activation of complement resulting in an immune
response directed against the nicotinic acetylcholine receptor(AchR) which leads, to impairement of neuromuscular transmission. Myasthenia gravis may occur in association with otherautoimmune diseases. Besides AChR muscle antibodies, withregards on serological immunopathology of Myasthenia Gravis,
antibodies such as against titin (1), and ryanodine receptor(5),are present in many sera from patients with thymic involvement
or late –onset Myasthenia. The impairment of neuromusculartransmission caused by AChR antibodies implies complementmediated focal muscle membrane damage (2), with accelerated
degradation of AChR (3).
Our goal in this study was to measure the levels of
AChR, C4, and IgG (because anti-AChR antibodies are Ig Gtype) in mysthenic sera and to correlate these concentrationin thymectomized and non-thymectomized M.G. patients
according to complement involvement, Ig G and anti-AChR
antibodies.
PATIENTS
Thirty (male/female: 26/4) MG patients were included.
Sixteen were thymectomized, three years ago and the rest
fourteen, were not thymectomized. Seven patients had additional
autoimmune disorders: three female patients had autoimmune

thyroiditis, another two female patients had systemic lupus erythematous, one female patient had chronic evolutive polyarthritis
and one male patient had autoimmune inflammatory myophaty.
All patients have received immunosuppressive therapy at thetime when blood samples were collected.
METHODSAll sera were stored at -80°C and at -20°C before
analysis,and treated in the same way. The same serum sampleof each patient was used for determination of AChR antibodies,
IgG and C4 complement component.
Sera were brought to room temperature prior to analysis
for C4 complement factor by laser nephelometric assay on a
Behring Nephelometry analyzer using rabbit polyclonal antiserum
to purified human C4, according with manufacturer instructions(Dade Behring).
Ig G was measured by the immunonephelometric determination using reagents of Dade Behring (Marburg GmbH). AChR
muscle auto-antibodies were measured by standard radioimmunoassay, using AChR, RRA kits (4).
Reference intervals for the parameters determined were thefollowing: 10-40 mg/dl - for C4 complement compound, 700-1600
mg/dl - for IgG, and < 0.4 nmol/l - for anti-AChR antibodies.
RESULTSFor the patients included in this study, we have performed
the immunoserological analysis including anti-AChR ab, C4
complement compound and IgG (Table I).

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

42

Fiziologia - Physiology  2010.20.2 (66)vals for the parameters determined were the following: 10-40 mg/dl - for C4, 700-1600 mg/dl - for IgG, and < 0.4 nmol/l - for anti-AChR antibodies.

s included in this study, we have performed the immunoserological analysisb, C4 complement compound and IgG (Table I).
Table I. Immunoserological values of thymectomized / non-thymectomized

unoserological values of thymectomized/ non-thymectomized patients
1600 with myasthenia gravis
patients with myasthenia gravis
Thymectomy
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No

AntiAChR ab(nmol/l)
126
250
19
120
50
9.5
125
42
22.47
12
16.98
11.33
274
25
134
211
0.4
0.4
50
9.5
0.4
0.4
0.4
0.2
4.25
0.4
0.21
0.1
0.8
0.5

C4
(mg/dl)IgG(mg/dl)

32
27
27
32
39
25
43
25
44
29
51
38
42
64
65
34
34
30
32
25
17
17
25
32
31
36
71
58
61
72

803
1419
998
954
1314
744
607
827
1781
828
831
701
1631
1210
884
872
912
747
666
744
874
905
1385
1111
1901
1515
1612
928
318
611

1400
1200
1000
800

16 non thymec tomiz ed
patients

600

14 thymec tomiz ed
patients

400
200
0
IgG -aA C hR

Fig. 2. Correlation between IgG and anti-AChR antibodies in myasthenic sera.
The error bars, represents mean ± SD
Thymectomy can induce a considerable reduction in IgG concentrations andan increase in level of C4 (Fig.3).
1400
1200
1000
800

16 non thymec tomiz ed
patients

600

14 thymec tomiz ed
patients

400
200
0
IgG -C 4

Fig.3. Correlation between IgG and C4 in myasthenia gravis sera. The error
bars,represents mean ± SD

The ratio IgG-ANTI-AChR ab/IgG-C4, show a lower level in
non-thymectomized patients, and a high level in thymectomized
this, we tried to see
the relationshipand the contried to see Except
the relationshipandthe concordance/correlation
betweenanti-AChR
patients due tothe other associate
autoimmune disease and tocordance/correlation between anti-AChR ab, C4 and IgG, in
the immunosuppressiveof thymectomized and non-thymectomized patients withmyasthenia therapy
gravis(Fig.4).
(Fig.1
sera of thymectomized and non-thymectomized patients withmyasthenia gravis (Fig.1 and 2).
1.11600 1600

1.08

1400 1400

1.06

1200 1200

1.04

1000 1000
800 800

thymec1616
nonnonthymectomiztomiz
ed edpatients
patients

600 600

thymectomiz
1414
thymectomiz
ed edpatients
patients

1

14 thymec tomiz ed
patients

0.98
0.96

400 400

0.94

200 200
0

16 non thymec tomiz ed
patients

1.02

0.92
(IgG -aA C hR )/(IgG -C 4)

0

aA C aAhRC hRC 4C 4

IgG
IgG

Fig.4. Ratio between IgG-anti –AchR antibodies and IgG-C4 in myasthenic

i-AChR antibodies,C4
IgG antibodies,C4
in myasthenic
forsera,thymectomized
and nonthymectomized
sera.Theerror bars,represents mean ± SD
Fig. 1. Relevanceandof anti-AChR
and IgG insera,
myasthenicforthymectomizedTheand nonthymectomized
patients. The error
bars, represents
patients.
error
bars, represents
mean
± SD
mean ± SD

2010.20.2(66)  Fiziologia - Physiology

43

CONCLUSIONS
The important immunological dyscrasia between anti-AChR
ab, IgG and C4 complement compound is due to the fact that all
patients taken in this study have received immunosuppressive
therapy and surgical treatment, which could significantly influence the humoral immune response.
The complement mediated cell lysis or phagocytosis could
play a role in the pathogenesis of myasthenia gravis.
The anti-AChR antibody titer does not correlate with clinicalcourse of the disease. The detection of anti-AChR antibodies
helped us to establish the type of myasthenia: sero-negative or
sero-positive.
Other immunological elements exist, in order to moderate theimmune response in myasthenia gravis, such as: anti-MuSK ab,
CIC, anti-striational antibodies, and anti-RyR antibodies.
Acknowledgements
We thank Răzvan Tănase for his moral support and helping
hand.
REFERENCES

1. Aarli JA, Stefansson K, Marton LSG, Wollmann RL. Patients withmiastenia gravis and thymoma have in their sera IgG autoantibodies

against titin. Clin. Exp. Immunol. 1990; 82: 284-288.
2. Barohn RJ, Brey RL. Soluble terminal complement compounds inhuman myasthenia gravis. Clin. Neurology and Neurosurgery 1993;
95(4): 285-90.
3. Lefvert AK, Bergstrom K, Matell G, Osterman PO, Pirskanen R. Determination of acetylcholine receptor antibody in myastenia gravis –clinical usefulness and pathogenic implications. J Neurol. Neurosurg.
Psychiatry 1978; 41: 394403.
4. Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD.
Antibody to acetylcholine receptor in myastenia gravis: prevalence, clinical correlates, and diagnostic value. Neurology 1998; 51: 933-939.
5. Mygland A, Tysnes OB, Matre R, Volpe P, Aarli JA, Gihus NE. Ryanodine receptor autoantibodies in myastenia gravis patients with thymoma. Ann. Neurol. 1992; 32: 589-591.
Abbreviations and defining terms
Anti-AChR ab. Anti-acetylcholine receptors antibodies
Anti-MuSK ab. Anti muscle specific tyrosin kinase antibodies
Anti RyR ab. Anti-Ryanodine receptor antibodies
CIC. Circulating immune complexes
C4. Compound of complement system
IgG. Immunoglobulin G
SD. Standard deviation

PATOLOGIA IMUNOSEROLOGICA A PACIENTILOR CU MIASTENIA GRAVISREZUMAT
Anticorpii anti-receptor pentru aceticolina (AChR), sunt prezenti in serul a 80-90% dintre pacientii cu forma generalizata a Miasteniei
Gravis. Pe langa anticorpii anti-receptor pentru acetilcolina, in patologia imunoserologica a miasteniei sunt incriminati si alti anticorpi
precum anticorpii anti-tirozinkinaza muscular specifica (MuSK), anticorpi anti-ryanodina (RyR), anticorpi anti-fibra musculara striata.
Acesti anticorpi sunt capabili de a activa si sistemul complement. In serul pacientilor cu miastenie, componentele C3 si C4 ale
sistemului complement sunt detectabile de asemenea in concentratii variabile, depinzand de medicatie si de evolutia clinica a bolii.
Miastenia Gravis este tratata cu inhibitori ai colinesterazei si/sau cu medicatie imunosupresiva. Extirparea timusului (timectomia),poate avea ca rezultat remisiunea permanenta a bolii sau reducerea dozelor tratamentului medicamentos. Acesti factori, anterior
mentionati, pot modifica intr-un mod semnificativ profilul imunoserologic al pacientilor cu miastenie, permitand in acelasi timp o bunamonitorizare a bolii.
Cuvinte cheie: miastenia gravis, teste serologice, markeri imuni

44

Fiziologia - Physiology  2010.20.2 (66)