Official Journal of the Romanian Society of Physiological Sciences

CHIEF EDITOR
CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
FLORINA BOJINGABRIELA TANASIEDACIANA NISTORCALIN MUNTEANEDITORIAL BOARD
ARDELEAN AURELBADIU GHEORGHEBĂDĂRĂU ANCA
BENEDEK GYÖRGYBENGA GHEORGHEBUNU CARMEN
COJOCARU MANOLECUPARENCU BARBUCONSTANTIN NICOLAE
HAULICĂ ION
IANCĂU MARIAMIHALAŞ GEORGETA
MUNTEAN DANINAMUREŞAN ADRIANANESTIANU VALERIUOPREA TUDOR(Arad)(Constanţa)(Bucureşti)(Szeged)(Cluj)(Timişoara)(Bucureşti)(Oradea)(Bucureşti)(Iaşi)(Craiova)(Timişoara)(Timişoara)(Cluj)(Craiova)(New Mexico)PĂUNESCU VIRGILPETROIU ANA
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RÁCZ OLIVERRIGA DANSABĂU MARIUS
SIMIONESCU MAIA
SIMON ZENOSAULEA I. AURELSWYNGHEDAUW BERNARDTANGUAY M. ROBERTTATU FABIAN ROMULUS
VLAD AURELIANVOICU VICTORZĂGREAN LEON

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Official Journal of the Romanian Society of Physiological Sciences

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CONTENTS
1. REVIEW - The History of HLA and Its Related Diseases ..........................................................................................................................................................................................4
Elena Gai, Alexandra Gruia, Mirabela Cristea, Margit Serban, Smaranda Arghirescu, Cristian Jinca, Loredana Balint Gib, Valentin Ordodi, Calin Tatu, Victor Dumitrascu, Gabriela
Tanasie, Virgil Paunescu
2. REVIEW - Small Interfering RNAs and the RNA Interference Phenomenon ...........................................................................................................................................................9
Alexandra Boleman, Mirabela I. Cristea, Alexandra Gruia, Simona Anghel, Gabriela Tănăsie, Carmen Bunu, Virgil Păunescu
3. CASE REPORT - ACL (Anterior Cruciate Ligament) Reconstruction Using Hamstring Tendon Graft ......................................................................................................................14
Alina Şişu, Ciprian Cebzan, Codruţa Petrescu, Andrei Motoc, Daniela Cipu, Carmen Tatu, Fabian Tatu
4. Interrelation between Diabetes Mellitus -Thyroid Diseases ................................................................................................................................................................................17
Adriana Gherbon, Romulus Timar, Monica Mărăzan, Iulian Velea, Ioana Zosin
5. The Eﬀect of Epilepsy on the Age-Related Prolongation of the P300 Wave .........................................................................................................................................................24
Major Zoltán Zsigmond, Buzoianu Anca Dana, Perju-Dumbravă Lăcrămioara, Mărginean Ioan, Krausz Lajos Tibor, Văcăraş Vitalie, Brusturean-Bota Ema
6. Oxidants-Antioxidants Balance in Oral Lichen Planus .........................................................................................................................................................................................28
Ioana Scrobota, Teodora Mocan, Adriana Filip, Doina Daicoviciu, Grigore Baciut
7. Exhaled NO and Risk for Asthma in Patients with Allergic Rhinitis......................................................................................................................................................................32
Ioana Adriana Bujor, Ioana Corina Bocşan, Diana Deleanu, Victor Cristea
8. DRB1 Allele Frequency in a Population Group from the Western Part of Romania ..............................................................................................................................................36
Elena Gai, Alexandra Gruia, Florina Boldeanu, Mirabela Cristea, Diana Lungeanu, Margit Serban, Smaranda Arghirescu, Cristian Jinca, Loredana Balint Gib, Valentin Ordodi, Victor
Dumitrascu, Calin Tatu, Virgil Paunescu
9. LETTER TO THE EDITOR: Radioprotective Properties of Water with Low Content of Stable Isotopes: Critical Evaluation .......................................................................................39
Sergei V. Jargin

CUPRINS
1. REVIEW - Istoricul HLA si afectiunile asociate .......................................................................................................................................................................................................4
Elena Gai, Alexandra Gruia, Mirabela Cristea, Margit Serban, Smaranda Arghirescu, Cristian Jinca, Loredana Balint Gib, Valentin Ordodi, Calin Tatu, Victor Dumitrascu, Gabriela
Tanasie, Virgil Paunescu
2. REVIEW - ARN-uri mici de interferenta si fenomenul ARN interferentei ...............................................................................................................................................................9
Alexandra Boleman, Mirabela I. Cristea, Alexandra Gruia, Simona Anghel, Gabriela Tănăsie, Carmen Bunu, Virgil Păunescu
3. CASE REPORT - Reconstructia ligamanetului incrucisat lateral folosind grefon tendinos ...................................................................................................................................14
Alina Şişu, Ciprian Cebzan, Codruţa Petrescu, Andrei Motoc, Daniela Cipu, Carmen Tatu, Fabian Tatu
4. Interrelatiile dintre diabetul zaharat si afectiunile tiroidiene..............................................................................................................................................................................17
Adriana Gherbon, Romulus Timar, Monica Mărăzan, Iulian Velea, Ioana Zosin
5. Efectul epilepsiei asupra prelungirii duratei undei P300 indusa de varsta ..........................................................................................................................................................24
Major Zoltán Zsigmond, Buzoianu Anca Dana, Perju-Dumbravă Lăcrămioara, Mărginean Ioan, Krausz Lajos Tibor, Văcăraş Vitalie, Brusturean-Bota Ema
6. Balanta oxidanti-antioxidanti in lichenul plan oral.............................................................................................................................................................................................28
Ioana Scrobota, Teodora Mocan, Adriana Filip, Doina Daicoviciu, Grigore Baciut
7. Oxidul nitric in aerul expirat si riscul dezvoltarii astmului la pacientii cu rinita alergica ......................................................................................................................................32
Ioana Adriana Bujor, Ioana Corina Bocşan, Diana Deleanu, Victor Cristea
8. Frecventa alelei DRB1 in grupul populational din partea de vest a Romaniei .....................................................................................................................................................36
Elena Gai, Alexandra Gruia, Florina Boldeanu, Mirabela Cristea, Diana Lungeanu, Margit Serban, Smaranda Arghirescu, Cristian Jinca, Loredana Balint Gib, Valentin Ordodi, Victor
Dumitrascu, Calin Tatu, Virgil Paunescu
9. LETTER TO THE EDITOR: Proprietatile radioprotectoare ale apei cu continut scazut de izotopi stabili: evaluare critica .........................................................................................39
Sergei V. Jargin

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REVIEWTHE HISTORY OF HLA AND ITS RELATED DISEASESELENA GAI1, ALEXANDRA GRUIA1, MIRABELA CRISTEA1, MARGIT SERBAN3, SMARANDAARGHIRESCU3, CRISTIAN JINCA3, LOREDANA BALINT GIB4, VALENTIN ORDODI5, CALINTATU1,5, VICTOR DUMITRASCU6, GABRIELA TANASIE1,2, VIRGIL PAUNESCU2
1
Center for Transplant Immunology, Emergency Clinical County Hospital Timisoara, Romania
2
Department of Physiology-Immunology, University of Medicine and Pharmacy “Victor Babes”
3
Pediatrics Department, University of Medicine and Pharmacy “Victor Babes” Timisoara
4
Stem Cell Transplantation Department, Emergency Clinical Children Hospital “Louis Turcanu”, Timisoara, Romania
5
Department of Biology, University of Medicine and Pharmacy “Victor Babes” Timisoara
6
Central Laboratory, Emergency Clinical County Hospital Timisoara, Romania

INTRODUCTION
Allogeneic tumor transplantation was often used in earlyexperiments to study tumor biology. In the early 1900s, two US
geneticists, Ernest E. Tyzzer and Clarence C. Little, performedsome crucial tumor transplantations in the offspring of crosses
between mice that were susceptible or resistant to an allogeneic
tumor. An antigen responsible for rejection was first discovered by
the British physician and pathologist Peter A. Gorer in 1936, working at that time in the Lister Institute for Preventive Medicine in
London. Following a suggestion from the British geneticist J.B.S.
Haldane, he studied whether resistance factors to the growth ofallogeneic tumors might be associated with some blood groupantigens. First, he found that his own serum contained ‘natural’
antibodies that could distinguish between erythrocytes of three
inbred strains of mice (1). Snell was studying tumor resistancegenes, which he called histocompatibility or H genes.
Three papers appeared in 1958 by Jean Dausset, Jon van
Rood and Rose Payne and their associates, respectively (2-4),
which laid the foundation of what was later to become the description of the first human leukocyte antigens – the HLA complex. The
discovery of the major histocompatibility antigens as so-calledstrong transplantation antigens, which play a major role in graft
rejection after transplantation, has its origin in the animal experimental analysis of tissue transplantations with mice.
In 1960, Payne, together with Julia and Walter Bodmer,
also using sera from multiparous women, not only detected two
leucocyte antigens, LA1 (later HLA-A1) and LA2 (later HLA-A2),
apparently controlled by alleles but also postulated at least one
additional antigen, LA3, determined by an additional allele atthe same locus (5).
A reversal point in the history of tissue typing was the firstWorkshop and Conference on Histocompatibility, which wasorganized by Bernhard Amos (Durham, NC) in 1964. During
this workshop the participants compared the reactivity of theirsera with various techniques. The results were incompatible,and could not be published.
During the second Workshop, which took place in 1965 in
Leiden, the results were more consistent. Hereon Paul Terasaki
and John McClelland at UCLA introduced the complement-

dependent microlymphocytoxicity technique. This method has
remained the standard serological test for HLA typing.
Sheehy et al. (6) reported that LD (or Dw) determinants
could also be identified by priming lymphocytes against given
LD determinants, which was called primed LD typing. Later
studies showed that the provisional HLA-D locus consisted ofseveral different closely linked loci, which encoded three different
series of determinants, DR, DQ (previously called DC) and DP(previously called SB).
In 1968, an HLA Nomenclature Committee was established(sponsored by World Health Organization, (WHO)) consistingof leading investigators in the field. This committee, which still
exists, is responsible for giving official names to HLA specificities and loci.
Kissmeyer-Nielsen and associates established that there
were two HLA loci (7). They called the two loci ‘LA’ (adaptedfrom the LA antigens of Payne and coworkers) and ‘4’ or ‘four’(adapted from the 4a and 4b antigens of van Rood).
In the early 1980s, the overall picture was that the HLA
chromosomal region, found to be present on the short arm ofchromosome number 6, encoded six different very polymorphic
series of determinants, A, B and C that were present on mostnucleated cells and DR, DQ and DP that remained present on
B cells, monocytes and dendritic cells.
In 1967, Ceppellini had introduced the term HLA haplotype
for the genetic information carried by each of the two HLA
chromosomal regions of an individual. Klein (8) introduced theterms class I to describe the A, B and C antigens and class II todescribe the DR, DQ and DP antigens (and the corresponding
antigens in other species), a nomenclature that has since been
followed. Furthermore, after the discovery of additional class I
antigens, HLA-G, -E and -F, with a more limited tissue distribution, the latter were named non-classical HLA class I antigens,while the HLA-A, -B and -C antigens were named classical HLAclass I antigens.
NOMENCLATURE OF HLA GENES AND ALLELES
The nomenclature of the HLA antigens is split by the information content of immunological methods (binding of antibodies,

Received September 2010. Accepted October 2010. Address for correspondence: Elena Gai, Center for Transplant Immunology, Emergency Clinical County Hospital
Timisoara, Romania, e-mail: elagai@yahoo.com

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cellular methods) and DNA based typing methods. A number of
HLA gene fragments have been reported and named. These
are HLA-T previously known as HLA-16, HLA-U previously
known as HLA-21, HLA-V previously known as HLA-75, HLAW, HLA-80, HLA-P, HLA-90, HLA-Y, and HLA-BEL/COQ/DEL
respectively (10).
a. Conditions for acceptance of new allele sequences (10)As emphasized in previous reports, there are required conditions for acceptance of new sequences for official names.
1. Where a sequence is obtained from cDNA, or where PCR
products are sub-cloned prior to sequencing, several clones
should have been sequenced.
2. If direct sequencing of PCR amplified material is performed, products from at least two separate PCR reactions musthave been sequenced.
3. In individuals who are heterozygous for a locus, andwhere one of the alleles is novel, the novel allele must be sequenced in isolation from the second allele.
4. A novel sequence should be confirmed by typing of genomic DNA using PCR-SSOP or PCR-SSP method. Where a
new sequence contains either a novel mutation or a previously
unseen combination of nucleotides, this must be confirmed by
a DNA typing technique.
5. An accession number in a databank should have beenobtained. Sequences may be submitted to the databases online
at the following addresses:
EMBL: www.ebi.ac.uk/Submissions/index.html
GenBank: www.ncbi.nlm.nih.gov/Genbank/submit.html
DDBJ: www.ddbj.nig.ac.jp/sub-e.html
6. Full-length sequences are preferable though not essential; the minimum requirements are complete exons 2 and3 for an HLA class I sequence and complete exon 2 for an HLAclass II sequence.
7. Where a novel sequence differs only within an intron orother non-coding part of the gene, a full-length sequence must
be obtained, which covers all coding and non-coding regions.
In the absence of a full-length genomic sequence from the mostclosely related allele that is identical in its exon sequence, it may
be required that this also be sequenced and submitted before aname can be assigned to the novel sequence.
8. Sequences derived solely from tumour material will not
be considered for nomenclature.
9. The complete HLA type for the HLA-A, -B and -DRB1
genes should be submitted for the material in which a novel
allele has been defined.
10. Submission of a sequence to the WHO Nomenclature
Committee should be performed using the online submission tool
available at www.ebi.ac.uk/imgt/hla/subs/submit.html.
A list of the newly reported alleles is published each monthin nomenclature updates in the journals Tissue Antigens, Human
Immunology and the International Journal of Immunogenetics.
The listing of references to new sequences does not imply priorityof publication (10).

b. New Allele Sequences
A total of 2558 HLA alleles have been named since the lastreport. For HLA class I, 616 HLA-A, 913 HLA-B, 446 HLA-C,
four HLA-E, 19 HLA-F, 31 HLA-G, 12 HLA-H, nine HLA-J, six
HLA-K, five HLA-L, four HLA-P and three HLA-V alleles werenamed, making a total of 3249 class I alleles with official names.
For HLA class II, 368 HLA-DRB1, 12 HLA-DRB3, one HLADRB4, one HLA-DRB5, seven HLA-DQA1, 45 HLADQB1, six
HLA-DPA1, 22 HLA-DPB1, one HLA-DMB and four HLA-DOA
alleles were named, making a total of 1198 class II alleles with
official names (10).
INTRODUCTION OF COLON DELIMITED HLA ALLELE
NAMESThe convention of using a four-digit code to distinguish HLA
alleles that differ in the proteins they encode was introduced in
the 1987 Nomenclature Report (11). Since that time additional
digits have been added, and currently an allele name may becomposed of four, six or eight digits dependent on its sequence.
The first two digits describe the allele family, which often corresponds to the serological antigen carried by the allotype. The thirdand fourth digits are assigned in the order in which the sequences
have been determined. Alleles whose numbers differ in the first
four digits must differ by one or more nucleotide substitutions thatchange the amino-acid sequence of the encoded protein. Alleles
that differ only by synonymous nucleotide substitutions within thecoding sequence are distinguished by the use of the fifth andsixth digits. Alleles that only differ by sequence polymorphisms
in introns or in the 5’ and 3’ untranslated regions that flank theexons and introns are distinguished by the use of the seventhand eight digits (10).
In 2002, the A*02 and B*15 allele families had more than 100
alleles (12). At that time the decision taken was to name further alleles in these families in the rollover allele families A*92 and B*95
respectively. For HLA-DPB1 alleles, it was decided to assign new
alleles within the existing system, hence once DPB1*9901 hadbeen assigned, the next allele would be assigned DPB1*0102,
followed by DPB1*0203, DPB1*0302 etc.
With the ever increasing number of HLA alleles described it
has been decided to introduce colons (:) into the allele names toact as delimiters of the separate fields. To facilitate the transition
from the old to the new nomenclature, a single leading zero must
be added to all fields containing the values 1 to 9 but beyond thatno leading zeros are allowed. This will help to lessen any confusion in the conversion to the new style of nomenclature (10).
Hence
A*01010101 becomes A*01:01:01:01
A*02010102L becomes A*02:01:01:02L
A*260101 becomes A*26:01:01
A*3301 becomes A*33:01
B*0808N becomes B*08:08N
DRB1*01010101 becomes DRB1*01:01:01:01
For allele families that have more than 100 alleles such as
the A*02 and B*15 groups it will be possible to encode these ina single series. Thus the A*92 and B*95 alleles have now been
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renamed in to the A*02 and B*15 allele series (10).
For example:
A*9201 becomes A*02:101
A*9202 becomes A*02:102
A*9203 becomes A*02:103 etc
B*9501 becomes B*15:101
B*9502 becomes B*15:102
B*9503 becomes B*15:103 etc
The names A*02:100 and B*15:100 will not be assigned (10).
In cases of other allele families where the number of allelesreaches 100 these will be numbered sequentially, for example
A*24:99 will be followed by A*24:100 (10).
The DPB1 allele names that have been previously assigned
names within the existing system have also be renamed, forexample (10):DPB1*0102 becomes DPB1*100:01
DPB1*0203 becomes DPB1*101:01
DPB1*0302 becomes DPB1*102:01
DPB1*0403 becomes DPB1*103:01
DPB1*0502 becomes DPB1*104:01 etc
The ‘w’ will be removed from the HLA-C allele names, butwill be retained in the HLA-C antigen names, to avoid confusion
with the factors of the complement system and epitopes on the
HLA-C molecule often termed C1 and C2 that act as ligands for
the Killer-cell Immunoglobulin-like Receptors (10).
Cw*0103 becomes C*01:03
Cw*020201 becomes C*02:02:01
Cw*07020101 becomes C*07:02:01:01 etc
These changes to the HLA Nomenclature were officiallyintroduced in April 2010. A full listing of old and new HLA allele
names was made available through the IMGT/HLA Database(www.ebi.ac.uk/imgt/hla) (13) and implemented with the April
2010 release of the database (10).
MOLECULAR STRUCTURE AND FUNCTION OF HLA
MOLECULES
The extended collection of genes on the short arm of
human chromosome 6 at 6p21.3 is called Major Histocompatibility Complex (MHC). This region is very gene dense andhighly polymorphic. It was originally identified and named
because of its role in tissue rejection after transplantation.
Furthermore, many of the genes in this region show an important role in the biology of the immune system. The MHC
is subdivided into three classes. The classification is basedon main functional characteristics of the genes within eachof the classes. The human MHC class I and class II are also
called HLA class I and class II. Genes in the HLA code for HLA
molecules. The main role of the HLA molecules has little to dowith transplantation. Originally these molecules are receptors,
which capture peptides or protein fragments of antigens. They
are present on cell surfaces where they can be recognized byappropriate T cells. With foreign antigens displaying to cells,
HLA molecules evoke cytotoxic T lymphocyte (CTL). CTL ishelper for a T cell response, which then regulates specificimmunity (14, 15).

Fig. 1. Schematic representation of a class I antigen

In human β2-microglobulin is a non-polymorphic protein,which is encoded on chromosome 15. It has the structure of an
immunoglobulin C domain. β2m is also associated with a numberof other class I-related molecules, for instance the products of
the CD1 genes on chromosome 1. The HLA-molecules of the
MHC class I region are expressed ubiquitously (14).
HLA class II
HLA class II molecules are membrane glycoprotein formed
through the non-covalent association of a 32 kDa α-chain and a
28 kDa β-chain. The α- and β-chains of all traditional HLA class
II molecules have the same overall structure (15).
The structure contains two extracellular domains, which arereferred to as α1-α2, and β1-β2, respectively. The domains distalof the membrane, α1 and β1 together form a peptide-binding
groove (Figure 2) (15).
Fiziologia - Physiology z 2010.20.4 (68)6cmyb

HLA class I
Telomeric region of the MHC have the genes of the MHC
class I. This class contains genes which encode the α-chainof HLA class I molecules. The region of the MHC class I
expand over 2 Mb. These genes are also members of theimmunoglobulin gene family. The molecules of the MHC class
I genes are involved in the presentation of peptides predominantly derived from intracellular proteins, to CD8+ cytotoxic
T-cells. The α-chains form heterodimers with the non-MHC
coded β2-microglobulin (β2m). The heavy α-chain (45 kDa)
is non-covalently associated with the β2-microglobulin (12kDa) a polypeptide which is also found free in serum. The
HLA class I α-chain contains three extracellular domains,
a transmembrane region and the cytoplasmic C-terminus.
The extracellular domains are α1, α2 and α3. The domains
α1 (N-terminal) and α2 together create the peptide-binding
groove (14).

6before SBT.

Fig. 2. Schematic representation of a class II antigen

HLA class II molecules have a more limited tissue distribution than HLA class I molecules. Class II molecules are mainly
expressed in the cells of the immune system such as B-cells, dendritic cells, macrophages and activated T-lymphocytes (15).
MHC class II is the most centromeric class of the MHC. This
class contains the genes HLA-DP, HLA-DQ and HLA-DR, as theso called classical MHC II genes, as well as HLA-DM and HLADO as non-classical MHC II genes. Genes of the MHC class II
are found in pairs, encoding the α- and β-chains, which form theheterodimers of the MHC class II protein molecules (15).
CURRENT METHODS FOR HLA TYPING
Since the mid-80s serological typing methods, which werecarried out exclusively (16), are complemented by analysis at the
DNA level. During the 9th IHCW in 1984, several papers described
the use of restriction fragment length polymorphisms method tostudy HLA at the DNA level (17). The introduction of the PCR (18)allowed the development of a variety of simple and rapid DNA
sequence based typing methods (19, 20). Nowadays several
methods, which describe the HLA at DNA level, are standard
methods in HLA typing laboratories. The most frequently usedmethods in the HLA typing laboratories are “Sequence Specific
Oligonucleotide Probe Hybridization” (SSOP), “Sequence SpecificPrimers Amplification” (SSP), “Reverse Line Blot” and “Sequence
Based Typing” (SBT). The latter method is in principle sequencingof the different HLA genes. It is the only technique which allows
identification of unknown HLA alleles immediately. Another highresolution technique which is used in quite a few HLA typing
laboratories is “Reference Strand Conformation Analysis”. All these
DNA based methods have in common the use of polymorphisms
in the DNA sequence to identify the HLA type.
Nevertheless serological typing methods are still standard
procedures in many HLA typing laboratories. For example,
many laboratories use serological typing as a pre-typing method

HLA ANTIGENS ARE ASSOCIATED WITH DISEASESThe human leukocyte antigen (HLA) region has been associated with hundreds of human diseases, including most of
autoimmune diseases (21). HLA-associated diseases can be theresult of the combination of different HLA molecules expressedat various loci (class-I and/or class-II) rather than the resultof one HLA variant only. Almost all HLA-associated diseases
are multifactorial polygenic diseases in which HLA allele(s), in
combination with other genetic variants and environmental factors, is involved in disease susceptibility. Human autoimmune
diseases are phenotypically heterogeneous, in terms of clinicalpresentation, age at onset, association with other autoimmune
disorders, and severity or rapidity of evolution, and it is likelythat different alleles, or allelic combinations at different loci, will
predispose to different forms of the disease (22).
The molecular mechanisms underlying HLA associations
have been determined in few autoimmune diseases in which
both autoantigen(s) and specific disease-associated HLA variants were unambiguously identified, such as celiac disease (CD)and type 1 diabetes (T1D). A number of hypotheses have been
proposed on the basis of comparison HLA alleles at the sequencelevel and theoretical, but in the absence of known autoantigen,no molecular or functional analysis has been able to confirm any
hypothesis. This is particularly well illustrated in two diseases– ankylosing spondylitis (AS) and narcolepsy – in which HLA
associations are very strong and perfectly defined at the alleliclevel, but the mechanisms underlying these associations are still
unknown (22). In 1973, Brewerton et al. (23) and Schlosstein
et al. (24) independently reported a very strong association
between HLA-B27 and ankylosing spondylitis. The studies foundthat 88%–96% of the patients carried HLA-B27 compared with
8%–4% of healthy controls, respectively. Later, the same year,
Jersild et al. (25) first reported a strong disease association toan HLA class II antigen. Multiple sclerosis (MS) was found to
be more strongly associated to a determinant as established by
MLC typing, LD-7a (later to become HLA-Dw2, now HLA-DR2),
than HLA-B7. The association of HLA-B27 with AS is one of thestrongest; 90–95% of patients with AS are positive for HLA-B27
compared with less than 10% of healthy subjects, which confers
B27 a high relative risk (>100). Given the disease prevalence(between 0.1% and 1.5%), the absolute risk of AS developing isabout 5% in HLA B27-positive individuals, and most HLA B27positive individuals remain healthy. Not all HLA-B27 allotypes
have a same predisposing effect: while B*2702, B*2704, andB*2705 (and probably B*2707) are associated with AS, B*2706
and B*2709 are not. It has been shown that the HLA-B*2705
and B*2709 subtypes (that differ in only a single amino acid)can bind a shared peptide with indistinguishable conformation,
whereas other peptides are bound in two extremely different
conformations and with different stability.
Narcolepsy is strongly positively associated with the DQB1*0602
allele and 90–100% of patients with definite cataplexy carry this allele. The closely related DQB1*0601 allele, which differs at only nine7

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residues, protects against developing narcolepsy, suggesting that
peptide-binding differences between these two alleles determine
whether they predispose to or protect against narcolepsy. Becauseof the strong DQB1*0602 association, narcolepsy has long beensuspected to be an autoimmune disease like virtually the other entire
major histocompatibility complex (MHC)-associated disorders.
Regarding celiac disease (CD) more than 90% of patients
express the HLA-DQ2 molecule, encoded by HLA-DQA1*0501DQB1*0201, and most of the remaining patients carry DQ8,
encoded by DQA1*0301-DQB1*0302. Thus, HLADQ2 and HLADQ8 are necessary, but there are not sufficient to predispose to
CD (most DQ2- or DQ8-positive individuals remain healthy). A
large number of non-HLA genes likely contribute to the pathogenesis of CD, but the participation of a single predisposing
non-HLA gene might be quite modest.
T1D is a multifactorial and polygenic disease, which affects
about 1% of the population. Approximately 50% of the genetic
risk of T1D can be attributed to HLA genes, and a large numberof studies have shown that specific alleles at the DRB1, DQA1,
and DQB1 loci are strongly associated with T1D.
CONCLUDING REMARKS
It is now more than 50 years since Dausset first discovereda leukocyte antigen in man, which became the first HLA antigen,
HLA-A2. Since then, the field has moved from histocompatibility
to become one of the most central fields in basic and clinical immunology in general. The term MHC for the HLA complex andsimilar genetic complexes in animals should be considered amisnomer because the role of the HLA class I and II molecules
as histocompatibility antigens is more a side-effect of their immunobiological function (25).
There are several reasons for the quick and extensive developments of the field. First are the instrumental contributions
by its many pioneers. Some of them have received the Nobel
Prize (Snell, Dausset, Benacerraf, Zinkernagel and Doherty).
That such a relatively large number of Nobel Prizes has gone topioneers in this field witnesses its importance. But another factor
that must not be underestimated is the extensive international
collaboration, which has taken place since the early days of HLA,in particular the IHWSs. Together, the pioneers and the extensive
international collaboration are responsible for the giant progress
we have seen in this field during the past 50 years (25).
Statistic studies proved that the frequency of an HLA antigen is abnormally increased in individuals with certain diseases(susceptibility genes) or decreased (protective genes) (26)
HLA-disease association may be generated by a connection which requires an intra-familial study, or a connection whichtranslates in a relative risk between the sick subjects and thenormal ones of the same population (26).
REFERENCES
1. Gorer P. The detection of a hereditary genetic difference in the blood of miceby means of human group A serum. J Genet, 1936, 32:17-31.
2. Dausset J. Iso-leuco-anticorps. Acta Haematol., 1958, 20:156-66.
3. van Rood JJ, Eernisse JG, van Leeuwen A. Leucocyte antibodies in sera

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cmybfrom pregnant women. Nature, 1958, 181:1735-6.
4. Payne R, Rolfs MR. Fetomaternal leukocyte incompatibility. J Clin Invest.,
1958, 37:1756-62.
5. Payne R, Tripp M, Weigle J, Bodmer W, Bodmer J. A new leukocyte isoantigen system in man. Cold Spring Harb Symp Quant Biol, 1964, 29:285-95.
6. Sheehy MJ, Sondel PM, Bach ML, Wank R, Bach FH. HL-A LD (lymphocyte defined) typing: a rapid assay with primed lymphocytes. Science, 1975,
188:1308-10.
7. Kissmeyer-Nielsen F, Svejgaard A, Hauge M. Genetics of the human HL-Atransplantation system. Nature, 1968, 219:1116-9.
8. Klein J. The Major Histocompatibility System in Man and Animals. Berlin:
Springer-Verlag, 1977.
9. Marsh SGE, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA,
Fernández-Viňa M, Geraghty DE, Holdsworth R, Hurley CK, Lau M, Lee KW,
Mach B, Maiers M, Mayr WR, Müller CR, Parham P, Petersdorf EW, Sasazuki
T, Strominger JL, Svejgaard A, Terasaki PI, Tiercy JM, Trowsdale J. Nomenclature for factors of the HLA system, Tissue Antigens, 2010, 75:291-455.
10. Bodmer WF, Albert E, Bodmer JG, Dausset J, Kissmeyer-Nielsen F, Mayr
W, Payne R, van Rood JJ, Trnka Z, Walford RL. Nomenclature for factors ofthe HLA system 1984. Vox Sang, 1985, 48(1):42-9.
11. Marsh SG, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, Geraghty DE, Hansen JA, Mach B, Mayr WR, Parham P, Petersdorf EW, Sasazuki
T, Schreuder GM, Strominger JL, Svejgaard A, Terasaki PI. Nomenclature forfactors of the HLA system, 2002. Tissue Antigens, 2002, 60:407-64.
12. Robinson J, Malik A, Parham P, Bodmer JG, Marsh SGE. IMGT/HLA
database – a sequence database for the human major histocompatibility
complex. Tissue Antigens, 2000, 55:280-7.
13. Rhodes DA, Trowsdale J. Genetics and molecular genetics of the MHC.
Reviews in Immunogenetics, 1999, 1: 21-31.
14. Nelson CA, Fremont DH. Structural principles of MHC class II antigen
presentation. Reviews in Immunogenetics, 1999, 1: 47-59.
15. Williams F, Mallon E, Middleton D. Development of PCR-SSOP for HLA-A
typing of bone marrow registry donors. Tissue Antigens, 1997, 49(1):61-6.
16. Brautbar C, Zlotogora J, Laufer N, Cohen T, Albert ED. Do identical HLADR3 genes convey susceptibility to celiac disease and insulin dependent
diabetes mellitus? Tissue Antigens, 1984, 23(1):58-60.
17. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N.
Enzymatic amplification of beta-globin genomic sequences and restriction siteanalysis for diagnosis of sickle cell anemia. Science, 1985, 230:1350-54.
18. Bodmer JG, Marsh SG, Albert ED, Bodmer WF, Dupont B, Erlich HA,
Mach B, Mayr WR, Parham P, Sasazuki T. Nomenclature for factors of the
HLA system, 1990. WHO Committee for Factors of the HLA System. European
Journal of Imunnogenetics, 1991, 18:33-55.
19. Erlich HA, Opelz G, Hansen J. HLA DNA typing and transplantation.
Immunity, 2001, 14:347-356.
20. Shiina T, Inoko H, Kulski JK. An update of the HLA genomic region,
locus information and disease associations: 2004. Tissue Antigens, 2004,
64:631-49.
21. Caillat-Zucman S. Molecular mechanisms of HLA association with autoimmune Diseases, Tissue Antigens, 2009, 73(1):1-8.
22. Brewerton DA, Caffrey M, Hart FD, James DCO, Nicholls A, Sturrock RD.
Ankylosing spondylitis and HL-A 27. Lancet, 1973, 1:904-7.
23. Schlosstein L, Terasaki PI, Bluestone R, Pearson CM. High associationof an HL-A antigen, W27, with ankylosing spondylitis. N Engl J Med, 1973,
288(14):704-6.
24. Jersild C, Fog T, Hansen GS, Thomsen M, Svejgaard A, Dupont B.
Histocompatibility determinants in multiple sclerosis, with special reference
to clinical course. Lancet, 1973, 2:1221-5.
25. Thorsby E. A short history of HLA. Tissue Antigens, 2009, 74:101–116.
26. Paunescu V, Homberg JC. Immunology, Ed. Orizonturi Universitare,
1999, pg.71-82.

8

REVIEW

SMALL INTERFERING RNAs AND THE RNA INTERFERENCE
PHENOMENONALEXANDRA BOLEMAN1, MIRABELA I. CRISTEA2, ALEXANDRA GRUIA2, SIMONAANGHEL2, GABRIELA TĂNĂSIE1,2, CARMEN BUNU1, VIRGIL PĂUNESCU1
1”Victor Babes” University of Medicine and Pharmacy Timisoara
2
Immunophysiology and Biotechnologies Center, Emergency Clinical County Hospital Timisoara, Romania

ABSTRACTThe RNA interference phenomenon’s applicability both in functional genomics and as a therapeutic tool for different disease including
cancer has gained increased attention during the last few years. As a result the siRNA technology has rapidly evolved and currently
several suppliers offer ready to use siRNA designs that target the mRNAs of a high number of genes. At the same time discovery ofa high number of miRNA involved both in normal developmental stages but also in different pathological processes have gained alot of interest in its practical use as a prognostic marker. This review presents, based on a rich documentation, the main aspects ofRNAi mechanism, miRNA and siRNA pathways and their therapeutic applicability in cancer.
Key words: RNA interference, miRNA, siRNA, cancer

1. RNA interference mechanism
RNA interference (RNAi) is a phenomenon within the livingorganisms that controls gene expression by post-transcriptional
gene silencing mechanism. The RNAi is an evolutionarily conserved gene silencing mechanism whereby small sequencesof extrinsic dsRNA or intrinsic microRNA inhibit complementarypost-transcriptional mRNA (35). The ancestral function of theRNAi system is generally agreed to have been immune defense
against exogenous genetic elements such as transposoms andviral genomes (8, 32).
First pointed out by Fire et. al. 1998, RNAi occurs througha series of steps involving generation of small interfering RNAs
fragments through the action of a specific RNAaseIII endonuclease. The small molecules resulted are able to mediate degradation of their complementary mRNA by association with a nuclease
complex called the RNA-induced silencing Complex (RISC)(49). The dsRNAs (double stranded RNA) can be endogenous(originating in the cell) as pri-microRNAs expressed from microRNAs encoded genes within the genome. When the dsRNA isexogenous (coming from infection by a virus with a RNA genome
or laboratory manipulations), the RNA is imported directly into
the cytoplasm and cleaved to short fragments so called smallinterfering RNAs (siRNA) by the enzyme Dicer (53).
The RNA interference pathway has become very useful inexperimental biology to study the function of genes both in vitro,in cell culture and in vivo, in animal model. Routinely doublestranded RNAs are synthesized with a sequence complementary
to a gene of interest and introduced into a cell culture or organism, where once recognized as exogenous genetic material
activates the RNAi pathway. Using this mechanism, a drastic
decrease in the expression of a targeted gene can be induced.
The RNAi mechanism may only partially abolish expression of
the target gene. For this reason the RNAi silencing using siRNAtechnique is sometimes referred as a “knockdown”, to makedistinguish from “knockout” procedures in which expression of agene is entirely eliminated (42). As methods of mediating the RNAinterference effect two types of molecules have been developed:
chemically syntethized small double stranded RNAs (siRNAs)and short hairpin RNAs (shRNAs).
siRNAsmiRNAsPotential
prognostic
biomarkers

EndogenousRNAinterference

Exogenous

Potential genesilencingmediatingcontrol

dsRNAsshRNAsFig.1 MiRNAs and siRNAs implication in RNA interference phenomenon
schematic representation

2. MicroRNAs (miRNAs)
MicroRNAs (miRNAs) are short endogenous RNA molecules that have been shown to regulate, together with theepigenetic mechanisms the genes expression in plants andanimals. Is being estimated that miRNAs could regulate 33%of the human genome (1). Since the discovery of the founding
members of the class, let-7 and lin-4 miRNAs in Caenorhabditis
elegans the number of discovered miRNA has considerablyincrease. Currently a total of 940 human, 590 mouse and 171
D. melanogaster miRNA sequences are already registered at
the miRNA registry web site (52) and the list is expanding withevery new miRNA identified.
Even if only a small number of miRNAs are known for their
biological functions they are considered to have important roles
in diverse regulatory pathways like: development timing, cells
differentiation and proliferation, and cell death (31). Abnormal
expression of miRNAs has been associated with different types

Received September 2010. Accepted October 2010. Address for correspondence: Boleman Alexandra, “Victor Babes” University of Medicine and Pharmacy Timisoara,
Etimie Murgu Square No. 2A, Timisoara 300041, e-mail: alexandraboleman@yahoo.com

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of cancers, and also, germline and somatic mutations in humanmiRNAs were recently identified (39).
The fast rate of miRNAs discovery requires a strict evidence ofthe miRNAs identity within the scientific community. In order to avoid
unintended overlap, it is important for researchers around the world
to have free access to an independent arbiter of gene names andpermanent up-to-date repository for published miRNA sequences.
3. MicroRNA pathway
Although the first miRNA was identified over ten years ago, it
is only recently that scientists have begun to understand the roleand diversity of these fascinating molecules (11). MicroRNAs aredouble stranded RNAs of 21-25 nucleotides derived from endogenously expressed transcripts with characteristic hairpin structures.
In humans, the majority of miRNAs (70%) are transcribed fromintrons and/or exons, and approximately 30% are located in intergenes regions (50). Bradley and colleagues showed that ~70%of mammalian miRNA genes are located in defined transcription
units. Interestingly, 117 miRNA genes were found in the introns inthe sense orientation. Of these 117 intronic miRNAs, 90 miRNAs
are in the introns of protein-coding genes, whereas 27 miRNAs
are in the introns of non-coding RNAs (ncRNAs).
The miRNA biogenesis pathway starts in nucleus with thetranscription of a primary miRNA (pri-miRNA) from a miRNA
coding gene. The pri-miRNA has a hairpin structure and contains
70 up to 100 nucleotides. The primary miRNA is process in the
nucleus by the ribonuclease Drosha to become miRNA precursor(pre-miRNA). After processing, the pre-miRNA is transported in thecytoplasm by exportin 5 where a second ribonuclease, Dicer, digests the pre-miRNAs resulting in a 21-25 nucleotide miRNA. Dicer
is a highly conserved protein that is found in almost all eukaryoticorganisms such as microorganisms, plants and animals (27). At
this stage, the miRNA binds the RNA-Induced Silencing Complex(RISC), and aligns with the mRNA. Based on the complementarylevel between the miRNA and the target sequence, the mRNAcan either be translational repressed (partial complementary) or
cleaved (identical complementary) (19). In Figure 2 is presented
the schematic representation of miRNA pathway.

4. siRNA pathway
The siRNA pathway is considered an evolutionarily conserved response triggered by an externally introduced double
stranded RNA (dsRNA). The dsRNA detected within the cell activates Dicer ribonuclease, which will cleave it into small interfering
RNAs (siRNA) that are approximately 21-23 nucleotides (19).
The siRNA is successively loaded into an RNA-Induced
Silencing Complex (RISC) which facilitates the separation of
the two strands and alignment of the siRNA with its appropriate
target mRNA. Based on siRNA complementarities to its target
mRNA will repress or cleave the miRNA. In figure 2 is presented
the siRNA biogenesis.
Scientists have used the siRNA pathway by artificiallyintroducing either dsRNA or siRNA designed to degrade targeted mRNAs. These synthetic silencing reagents are used
as molecular biology tools for novel gene identification, genefunctional analysis, and biological pathways screening (23). In
order to be able to synthesize a siRNA a number of parameters
have been suggested: selection of a target cDNA region 50-100
nucleotides, downstream of the start codon, selection of a 5’AA(N19)UU target mRNA sequence where N is any nucleotide,
50% G/C content in the target sequence, avoidance of 5’ or 3’
untranslated regions and high G-rich areas, and confirmationof exclusive target-specific sequences (15). Presently, custom
siRNA synthesis service is available through a number of companies, including Dharmacon, Qiagen, and Ambion (5).

shARN

shARN
Dicer

siARN

RISC
ComplexPrimiARN

miRNA/
mRNA

PremiARN
Exportin 5
Dicer
miARN

Fig. 2. MiRNA pathway

In order to succeed siRNA internalization within the cells
different approaches have been used like: electroporation (30),soaking in siRNAs (28), feeding bacteria carrying dsRNA (4),transfection with commercial reagents (15), and vector-based
strategies (26, 34). The vector-based strategies involve either
DNA or viral vector- mediated RNAi. In the case of DNA vector
based strategy, RNA Polymerase II or III promoters have been
incorporated into DNA vectors along with siRNA expression
cassettes (3, 34, 43, 45). These cassettes have included either
sense and antisense siRNA strands expressed from tandem
promoters or a short hairpin (shRNA) cassette whereby the two
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Fig. 3. siRNa biogenesis

RISC
ComplexDrosha+Pasha

siRNA/mRNA

10

siRNA strands are separated by a short spacer (12). Use of RNAPolymerase II promoters to generate shRNAs has the advantagethat they allow easier adaptation of inducible/repressible tissueor cell-specific siRNA expression (47).
The plasmid-based siRNA technique has a low efficiency. In
order to substitute this deficiency viral vectors have been developed. In several published papers adenoviral vectors and various
retroviral vectors (e.g. lentivirus-based) have proven effective
delivery systems in numerous cell types including non-cycling
cells, stem cells, and zygotes (26, 37, 42).
3. MiRNA/siRNAs applications in cancer
The extensive studies made during the last few years
have indicated that many miRNAs are associated with primary
human tumors, and more than 50% of human miRNAs genes
are located at genomic regions implicated in cancers, such ascommon breakpoint regions and fragile sites (3). Several miRNA
have been found to have increase expression in different typeof cancers. According to Heneghan 2010, in breast cancer havebeen observed two types of miRNA (miR-21, miR-29b-2) withincrease expression, while other three miRNA are observed
to have decreased expression (miR-125b, miR-145 miR-10b).
Similar situations where observed for different types of tumors
like ovarian, thyroidal, testicular and so on.
Phenotyping and molecular profiling of human cancers havegreatly enhanced the diagnosis and biological classification ofseveral tumors, in particular breast cancers where the miRNAtechnology has enhanced disease classification beyond singlegene markers (21).
A comprehensive screening of the breast cancer subclasses
via miRNA expression profiling could further characterize themolecular basis underlying these subtypes, perhaps define
more precise subsets of breast cancer, and provide opportunities
for the identification of novel targets that can be exploited fortargeted therapy. In the studies taken by Iorio et al. 2005, wereanalyzed 76 breast tumor and 34 normal specimens, and succeed to identified 29 miRNAs that were differentially expressedin breast cancer tissue compared to normal, and a further setof 15 miRNAs that could correctly discriminate between tumor
and normal.
Because of the high incidents of cancers disease there isgreat need for the identification of sensitive, reliable and acceptable markers of response to neoadjuvant and adjuvant therapies.
To date no work has been published on the role of circulating
miRNAs in breast cancer—an area where, if feasible, their use
as novel minimally invasive biomarkers would be an incredible
breakthrough in the management of this disease (21). In order
to be extensively used a biomarker must accomplish several
characteristics like: easily accessible, it can be sampled relatively
non-invasively, sensitive enough to detect early presence oftumors’ in almost all patients and absent or minimal in healthy
tumor-free individuals.
MiRNAs are considered to have enormous potential toserve as an ideal class of cancer biomarkers for the following
considerate: they expression profiles is tissue-specific and is

known to be aberrant in cancer; are stable molecules that are
well preserved after different modalities of sample preservation
were applied: formalin fixed, paraffin embedded tissues as well
as fresh snap frozen specimens.
siRNA pathways is consider to be an important gene silencing mediating technique. siRNAs constructs can be easily
synthesized to triggered mRNAs of chosen genes considered tobe responsible of different pathologic processes. As a result wasobserved 80-90% down-regulation of the target gene expression.
This approached produce a transient effect, the mRNA of thetarget gene was observed to return to normal after 3-7 days (44).
In order to be suitable as a therapeutic tool an effective delivery
mechanism to assure integration into target tissues remains amajor impediment for RNAi therapy before they can be tried
clinically (40). Different strategies have been elaborated andcomprise different delivery system including synthetic small RNA
molecules by complexing or covalently linking with lipids and/or
delivery proteins (16, 12). Besides nonspecific agent like cationicliposome and cholesterol (41,51), novel nanotechnology-based
conjungation of bacterial phage packaging RNA with therapeutic
molecules, antibody-protamine, etc, have been designed todeliver small RNA assembly to target cells (41,17). Conjugation
with homing signals for tissue/cell-type specific delivery has
recently progressed (29). All development in this aspect plus theresearch on local delivery may eventually expand the therapeutic
opportunities for miRNA targeting.
Currently, there are several biotechnology companies developing clinical applications of siRNA in various human diseases
like: solid tumors, hepatitis C, HIV, Spinal Cord Injury, Parkinson
and Huntington disease (20). The current testing is in preclinicalor phase 1 clinical stage. In addition of low production costs andease synthesis (compared with protein or antibody therapies),
data indicates that siRNA has favorable pharmacokinetic properties and can be delivered to a wide range of organs (2). However,blood stability, delivery, poor intracellular uptake, and nonspecificimmune stimulation still present significant challenges for thedevelopment of RNAi reagents for clinical use (24).
4. Methods for screening miRNA
The explosion of interest in miRNAs over the past few years
requires effective tools for detecting their presence, quantificationand functional analysis. High-throughput profiling techniquessuch as miRNA microarrays and bead-based miRNA flow cytometric approach have facilitated miRNA expression profiling (7).
According to Dharmacon 2005, TaqMan quantitative qRT-PCR
and sequencing miRNAs are the most widely used methods, for
the identification of new miRNAs, while microarray analysis is ahigh throughput method that typically only reveals the expressionof known miRNAs.
Because of miRNA exceptional stability within the visceraltissue efforts have been made to establish several techniques
for detection, preservation and quantification of circulate miRNA
within the bodily fluids (blood, urine, saliva, etc). Until now successfully results have been obtain by isolating small RNA fractionof size typical of miRNAs within the blood plasma. Mitchell et
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al., 2008 have made experiments in order to point out miRNAspresence in clinical samples of plasma and serum in a stable
form. They isolated total RNA from human plasma from healthy.
The RNA obtained was amplified using 18-24 nucleotides primers provided by Dharmacon and gel purified by electrophoresis
through denaturing polyacrilamide gel. They research might
be considered the foundation for the development of miRNAs
as a novel class of blood-based cancer biomarkers and raise
provocative questions regarding the mechanism of stability andpotential biological function of circulating miRNAs.
5. Future perspectives
Since its initial discovery in 1998 RNAi has brought a lot of
excitement and hope in the scientific community. The promiseof RNAi screening has attracted many researchers but theirpractical applications still require new technical expertise. Even
if it has been intensively studied within the last years there arestill many unknowns that remain to be revel within the next
years. Development of more efficient delivery methods andregulated tissue-specific or differentiation-dependent expression of siRNA/shRNA are critical issues for transgenic studies
and gene therapy.
Computer assisted sequence predictions and more
complex understanding of miRNA mechanisms will promote
the development of artificial miRNAs. The miRNA signaturein susceptible individuals including their expression profiles,
dynamics, and even miRNA target variants (single-nucleotide
polymorphisms) may eventually enable the miRNA-based
individual-specific therapy, as well as disease diagnosis andprognosis. In addition, siRNA/miRNA specific delivery to target
cell populations using approaches of nanobiotechnology has
begun and shows increased potential. With the development
in miRNA field, these tiny molecules might become invaluable tools for various areas of basic and applied research andeventually for therapeutic intervention (50).
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Acknowledgement
This paper was supported by EUROPEAN SOCIAL
FUND, The Operational Sectorial Program for Human Resource Development 2007 – 2013, project title: Cellular andmolecular biotechnologies for medical applications, FSE
POSDRU/89/1.5/S/60746

ARN-URI MICI DE INTERFERENTA SI FENOMENUL ARN INTERFERENTEIREZUMAT

Aplicabilitatea fenomenului ARN interferenţei atât în genetica funcţională cât şi în scop terapeutic pentru tratarea diferitelor tipuri deboli, inclusiv cancer se bucură de tot mai mare interes. Ca rezultat tehnologia siARN s-a dezvoltat rapid şi în prezent diferiţi furnizorioferă designuri siARN pentru unu număr mare de mARN aparţinând diferitelor gene ţintă. În acelaşi timp, identificarea unui număr
mare de miARN implicate atât în stadiile de dezvoltare normale, dar şi în diferite procese patologice se bucură de un interes ridicat
privind utilizarea acestora în practică ca şi marker de prognostic. Acest articol prezintă, pe baza unei documentaţii bogate, principlalele
aspecte ale mecanismului ARNi, calea de sinteză a miARN şi siARN şi aplicabilitatea terapeutica a acestora în cancer.
Cuvinte cheie: interferenta ARN, miRNA, siRNA, cancer

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ACL (ANTERIOR CRUCIATE LIGAMENT) RECONSTRUCTIONUSING HAMSTRING TENDON GRAFT - A CASE REPORTALINA ŞIŞU, 1CIPRIAN CEBZAN, 1CODRUŢA PETRESCU, 1ANDREI MOTOC, 2DANIELA
CIPU, 3CARMEN TATU, 4FABIAN TATU
1
Department of Anatomy and Embryology, University of Medicine and Pharmacy “V. Babes” Timisoara, 2Department of Radiology and Medical Imaging of County Hospital Timisoara, University of Medicine and Pharmacy “V. Babes” Timisoara
3
Department of Physiology, University of Medicine and Pharmacy “V. Babes” Timisoara
4
University of Medicine and Pharmacy “V. Babes” Timisoara, Clinic Military Hospital
1

ABSTRACTACL rupture is in the most cases a sporting accident, due to the patient feels pain in knee joint, accompanied by regional deformation, enlarging joint volume and experiencing partial functional impotence. In our case, both tests for the instability of the knee joint
were positive. After laboratory investigations consisting in sagittal radiography of the knee joint to rule out any bone lesions, MRI isdone, putting it accurate diagnosis of ACL injury, more precisely an ACL rupture. For therapeutic purpose is decided arthroscopy, ACL
reconstruction was done using semitendinosus muscle tendon. Functional recovery takes between 6-8 months, mainly consistingof physiotherapy. Evolution is very good.
Keywords: ACL, semitendinosus muscle, reconstruction, tendon.

INTRODUCTION
Seen as a stabilizer of the knee joint, anterior cruciate
ligament (ACL) is to limit the movement of the tibia in relation
to the femur from the posterior toward anterior and at the sametime to prevent an anterior movement of the tibia to the femur,
and limiting an internal rotation of tibia in relation to the femur..
In ACL rupture the ligament heads distance themselves from one
another and are not returning to the original site as might happen
to the other ligaments of the knee joint (tibial collateral ligamentand fibular collateral ligament), where surgical reconstruction
is necessary (1).
The causes of ACL rupture are represented in majority of
injuries sports: skiing, soccer, and so on.

II. Clinical examination dataIn our emergency department the patient had his right knee
joint with an increase of its volume, accusing a severe pain andfunctional impotence in the joint. The joint presented also a purple
eritema, the tegument being in the same time hot. Drawer sign(used to detect rupture of the cruciate ligaments in the knee joint,with the knee flexed to 900) was positive, as well as Lachman
test (the knee flexed to 30 0).
III. Laboratory data
Being a young patient without a history of pathological,
have made only a complete blood count (CBC) and coagulation
profile, which were normal, no setting of medical problems, both,anesthetic and intraoperator.

MATERIALS AND METHODS
I. Anamnesis
C.F. 27 years old, male, suffered a skiing accident by falling ,patient accused a right knee joint pain and felt a cracmentsensation because of ACL rupture. In an attempt to support his
weight on his right leg the patient found a great instability of thejoint. It is reported that the patient is clinically healthy, showing
no acute or chronic disease.

IV. Laboratory investigations
In the emergency was made an X-ray of right knee joint, but
no bone lesions were detected. Coupled with the clinical examination was suspected a ligament damage. It was recommended
to perform a MRI exam, ligament injuries due to objectivity andreliability with a diagnosis of over 90%. MRI in conjunction withclinical examination gives a diagnosis of certainty, supporting theindication for surgery in the arthroscopic right ACL reconstruction(Figures 1 and 2).

Received October 2010. Accepted November 2010. Address for correspondence: Assistant Professor Alina Sisu, MD, Department of Anatomy and Embryology,
University of Medicine and Pharmacy “V. Babes” Timisoara, phone: 0724361671, e-mail: alinasisu@gmail.com; alinasisu@umft.roFiziologia - Physiology z 2010.20.4 (68)

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Fig.3. Harvesting tendonsFig. 1. Sagittal MRI shows a broken ACL

Fig.4. Preparation autograft

Fig.5. Preparation autograftFig. 2. MRI, ACL is broken, picture shows an entrapment ligament stump

V. Treatment and evolution
Arthroscopic surgery is the correct treatment. Semitendinosus muscle tendon is taken from the pes anserinus level forthe autograft preparation (Figure 3). If semitendinosus muscle
tendon is not thick enough, minimum 7 mm, can be taken a
gracilis muscle tendon. In our case the patient with an athleticbuild, the semitendinosus muscle tendon was enough. It was
prepared an autograft having dimensions as 10 cm in lengthand 8 mm in diameter (Figures 4 and 5).

Surgical Technique
The patient is positioned on the operating table in supine
position with the knee at 90 º, right leg is painted with iodine
solution, is isolated with sterile fields. Right semitendinosus
muscle tendon has been harvested through an oblique incision
centered on right pes anserinus (2). The autograft was preparedon workbench at optimal length and thickness, femur and tibia
were being tunneled in order to place the autograft, taking toaccount its size and in a stable position with specific kit tools forligament reconstruction, in order to realize a quasi-anatomicalreconstruction of the anterior cruciate ligament (5). After position15

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ing the autograft, it was fitted with two femoral transfix and tibial
interference with resorbable screw (Figure 6).
Evolution is the favorable in most cases and our caseis no exception to the rule, considering the patient’s age(young one) and because there is no intraoperative events inconjunction with an appropriate program of functional recoveryof the right knee joint using kinetotherapy. Recovery for sport
performance lasts about 6 months, depending on the patient’s
functional ability to perform physical exercises.

Fig.6. Fixing autograft

DISCUSSION
Anterior cruciate ligament reconstruction is the only way

to save the function of the knee which suffered rupture of thisimportant anatomical structure (3). In classical literature ACL
rupture is considered “the beginning of the end of the knee.” In
the absence of function of this ligament the joint degradation
continues through joint menisci lesions, cartilage lesions, andfinally the gonarthrosis disease appearance, whose treatment
is only articular endoprosthetic replacement (4).
We believe that for physically active individuals with age
around 50-60; this type of ligament reconstruction is indicatedin the presence of ACL ruptures.
REFERENCES
1. Bryant AL, Clark RA, Pua YH. Morphology of hamstring torquetime curves following ACL injury and reconstruction: mechanisms andimplications. J Orthop Res. 2011 Jan 21. doi: 10.1002/jor.21306.
2. Li Z, Liu Y, Wang Z, Li Z, Wei M, Cai X, Zhu J, Zhongguo Xiu Fu,
Chong Jian, Wai Ke, Za Zhi. Arthroscopic reconstruction of anterior
cruciate ligament for single bundle rupture with hamstring autograft,2010 Nov; 24(11): 1315-7.
3. Romanini E, D’angelo F, De Masi S, Adriani E, Magaletti M, Lacorte E, Laricchiuta P, Sagliocca L, Morciano C, Mele A. Graft selectionin arthroscopic anterior cruciate ligament reconstruction. J Orthop
Traumatol., 2010; 11(4):211-9.
4. Czamara A. Functional benchmarking of rehabilitation outcomes
following anterior cruciate ligament reconstruction. Ortop Traumatol
Rehabil., 2010;12(6):519-33.
5. Rhee PC, Levy BA, Stuart MJ, Thoreson A, An KN, Dahm DL.
A biomechanical comparison of the Delta screw and RetroScrew
tibial fixation on initial intra-articular graft tension. Knee Surg Sports
Traumatol Arthrosc., 2011.

RECONSTRUCTIA LIGAMENTULUI INCRUCISAT LATERAL (ACL)UTILIZAND GREFON TENDINOSREZUMAT
Ruptura ACL este de cele mai multe ori rezultatul accidentelor sportive, pacientul prezentand durere la nivelul articulatiei genunchiului,
insotita de deformare articulara, edem si impotenta functionala partiala. In cazul de fata, ambele teste efectuate pentru instabilitatea
articulatiei genunchiului au fost positive. Dupa efectuarea investigatiilor de laborator, care au constat in radiografii sagitale ale articulatiei genunchiului, pentru excluderea afectarii osoase, s-a efectuat RMN, in urma caruia a fost stabilit diagnosticul cu precizie,
si anume lezarea ACL – ruptura ACL. In scop terapeutic s-a decis efectuarea artroscopiei, iar reconstructia ACL a fost efectuataprin utilizarea tendonului muschiului semitendinos. Refacerea functionala dureaza intre 6-8 luni si consta in proceduri fizioterapice.
Evolutia post operatorie este favorabila.
Keywords: ACL, muschi semitendinos, reconstructie, tendon

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INTERRELATION BETWEEN DIABETES MELLITUS -THYROIDDISEASESADRIANA GHERBON1, ROMULUS TIMAR2, MONICA MĂRĂZAN3, IULIAN VELEA4, IOANAZOSIN51
Department of Physiology, UMF „V. Babeş”, Timişoara
2
Department of Internal Medicine III, County Hospital Timişoara
3
Department of Paediatrics, Hospital L.Ţurcanu Timişoara
4
Department of Paediatrics, Hospital Bega Timişoara
5
Department of Endocrinology, Clinical Emergency County Hospital Timişoara

ABSTRACT
Diabetes mellitus and thyroid diseases represents two endocrinopathies frecquently in general population. Because insulin and thyroid
hormones are implicated in celular methabolism, excess or deficiency of one of these determinated functional disorders of another.
The purpose of this study is to determinate influence of thyroid diseases on diabetes mellitus or influence of diabetes mellitus on
associated thyroid diseases. The studied group was of 733 cases, with an age between 7-79 years.
The studied group was subdivided according age in 2 subgroups: children subgroup and adults subgroup. We used clinical, imagistic,
biochemistry, hormones, imunological parameters.
Keywords: diabetes mellitus, thyroid diseases, interrelations

INTRODUCTION
Influence of thyroid disease on diabetes mellitus
Diabetes mellitus (DM) and thyroid disorders are two endocrinopathies frequently in the general population. Since insulin andthyroid hormones are involved in cellular metabolism, excess ordeficit of one of them determines the other functional disorders.
Thyroid disorders are commonly associated with DM in oldage, especially in type 2 diabetes, and with autoimmune diseases
in type 1 diabetes (9). Thyroid disorders complicate diabetes
treatment and diagnosis of diabetes complications. For example,
6.6% of the general population has thyroid disease, compared with
10.8% and 13.4% of people with diabetes. The high prevalenceoccurs in women with type 1 diabetes (because of the autoimmune
DM type) and because thyroid disorders are more common in
women. Also, postpartum thyroid disorders are three times morecommon in women with diabetes than in non-diabetic (5).
In terms of clinical, thyroid disorders may influence glycemicbalance. For example, hyperthyroidism can worsen glycemicbalance and increase insulin requirement. Also, thyrotoxicosis
can mask a subclinical DM.
A number of studies (16) agree with the following aspects:-hyperthyroidism may determinate worsening of glycemic
control-hyperglycemia may improve under thyrotoxicosis treatment-uunexplained worsening hyperglycemia may be due touncompensated hyperthyroidism
Graves-Basedow disease is the most common cause of
hyperthyroidism. While Graves-Basedow disease may be asso-

ciated with type 1 diabetes in the poliglandular autoimmune syndrome, thyrotoxicosis itself is diabetogenic. Glucose intolerance
occurs in 50% of patients with Graves-Basedow disease andclinical diabetes only at 2-3%. In patients with known diabetes
causes worsening glycemic control (3).
Disturbances that occur as a result of hyperthyroidism andcontribute to the deterioration of glycemic control are:
 Gastro-intestinal system. In hyperthyroidism occurs
accelerating gastric emptying, increase intestinal absorption of
glucose and increase portal venous flow (6).
 Insulin secretion. While some studies show decreased
insulin secretion in hyperthyroidism (3), most studies report a
normal or increased insulin secretion in peripheral blood andportal (3). It is possible that the exacerbation of insulin secretionis masked by increased insulin degradation. In hyperthyroidism,
insulin clearance rate is increased about 40% (3). Thyrotoxicosis
causes long-term beta-cell dysfunction, characterized by reduced
secretion of pancreatic insulin, inadequate insulin response to
glucose and decrease insulin secretion rate (3).
 Endogenous glucose production. In hyperthyroidism,
endogenous glucose production is increased by various mechanisms:- increasing the availability of gluconeogenetic precursors
by lactate, glutamine and alanine form in skeletal muscle andby glycerol in adipose tissue- elevated serum free fatty acids (AF) which stimulates
gluconeogenesis- increase in glycogenolysis caused by inhibition of glycogensynthesis

Received June 2010. Accepted July 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

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- increased expression of glucose transporter protein- increased secretion and exaggerated effects of glucagon
and adrenaline on liver cells (14)
Use of glucose
Adipose tissue
In adipocytes isolated from rats or from patients with hyperthyroidism, sensitivity of glucose transport and insulin use isnormal, increased or decreased (14).
Skeletal muscle
In skeletal muscle, there is an increased intake of glucose
and lactate formation. This occurs from stimulation of basal andstimulates insulin by the transporters GLUT-1 and GLUT-4,
increased glycogenolysis responsiveness to beta-adrenergic
stimulation, increased hexokinase and 5-fosfofructokinase activity
and decreased sensitivity glycogen synthesis to insulin (14).
Influence of diabetes on associated thyroid diseases
Malignant ophthalmopathy induced blindness may be
the most feared complication of Graves-Basedow disease. Is
determined by optic nerve compression through increasing
exrtraocular muscle at the orbital apex. Its incidence inpatients with diabetes is increased approximately 10 times.
High prevalence is explained by the reduction of optic nerve
oxygenation in diabetic patients because of vasculopathy (16).
When diabetes occurs in euthyroid subjects, causes alteredthyroid function tests, but not clinically manifest disorders. In
patients with preexisting ophthalmopathy of Graves’ disease,
the risk of vision loss is increased and recovery of sight islimited (16).
MATERIAL AND METHODS
Investigated population
The study included subjects with diabetes mellitus which
in time present thyroid disease or subjects with thyroid disease
who subsequently present diabetes mellitus
The study group comprised 733 cases aged 7-79 years.
Subjects were divided as follows: group of children that included 83 children and adolescents aged 7-17 years (14.57 ± 2.25 years), with a ratio F / M
of 5.9 / 1.
 group of adults that included 650 adults aged 18-79 years(52.03 ± 12.46 years), with a ratio F / M of 9.48 / 1.
Methods of investigation
Methods of investigation were the clinical data - history,
present status, imaging - ultrasound thyroid, biochemistry - carbohydrate metabolism parameters: fasting glycemia, glycosuria,
glycosylated hemoglobin and thyroid hormone investigations andsome immunological parameters.
Glucose determination was performed by enzymatic techniques with glucozooxidase. Were considered normal fastingblood glucose between 70-110 mg%, diabetes mellitus - fastingblood glucose values above 126 mg%, impaired glucose tolerance - fasting glucose values between 110-126 mg% and oral
glucose tolerance test (OGTT) at 2 h between 140-200 mg%

RESULTS AND DISCUSSION
The group of children and adolescents was represented by
83 subjects, aged 7-17 years (Table I). All children take in the
study had type 1 diabetes.
Table I. Distribution by age and sex of children and adolescents group

Age

Number ofcasesnFemale

%

n

Male

%

n

%

0 - 4 years

-

-

-

-

-

-

5 - 9 years

2

2.4

2

100

-

-

10 – 14 years

32

38.56

22

68.75

10

31.25

15 – 17 years

49

59.04

47

95.92

2

4.08

Fiziologia - Physiology z 2010.20.4 (68)

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and fasting impaired glucose tolerance - fasting glucose values
between 110-126 mg% and OGTT at 2 h under 140 mg%.
Determination of glycated hemoglobin (HbA1c) was achieved
through the DiaStat program for glycated hemoglobin HbA1c who
measures the ratio of glycated hemoglobin to total HbA.
Determination of serum levels of TSH, free fraction of triiodothyronine (FT3), free fraction of thyroxin (FT4) were ARCHITECT quantitative method, which is an imunodetermination bychemiluminescence’s with small Chemilumnescent Microparticle
Immunoassay (CMIA). The following values were considered
normal: TSH = 0.465 to 4.68 mIU / ml, FT3 = 3.69 -10.4 pmol /l, FT4 = 10 to 28.2 pmol / l.
Immunological parameters were represented by some markers of thyroid autoimmunity - antiTPO and antiTg antibodies.
To determine the serum titers Atc antiTPO Axsym antiTPO
kit was used, the method is enzyme immunoassay with micro
particles, Meia (Microparticle Enzyme Immunoassay). Was
considered normal: Atc. antiTPO (<35 IU / ml).
To determine the serum titers Atc antiTg Axsym antiTg
kit was used, the method is enzyme immunoassay with micro
particles, Meia (Microparticle Enzyme Immunoassay). It wasconsidered normal: Atc. antiTg (<55 IU / ml).
Thyroid ultrasound performed in all cases is a non-invasive
method of exploration that allows measurement of thyroid volume, thyroid study report with cervical anatomical structures,
thyroid parenchyma changes etc.
The appearance of normal thyroid parenchyma is characterized by a high intensity echogenic, homogeneous, easily distinguishable from the neck muscles which look hypoecogenic.
Inflammatory and autoimmune processes are hypoecogenic. Degree of thyroid hypoecogenity was assessed as: discreet+, moderate + + and marked + + +.
In autoimmune thyroid disease is found hipoecogenity ofthyroid parenchyma.
Graves’ disease appears: thyroid volume generally increased and hipoecogenity with different intensities with variablehomogeneity.
Chronic autoimmune thyroiditis appears: hipoecogenity
generally uneven and normal or increased thyroid volume.

18

Regarding the type of thyroid disease, the first priority wasin the ACT, followed by diffuse goiter with euthyroidism andGraves-Basedow disease (Table II).
Adult group consisted of 650 people, young adults andelderly adults, aged between 17 and 79 years (Table III).
Table II. Distribution by type of thyroid disease in the lot of children
and adolescentsTypeof thyroid
diseaseACT
Diffusegoiter witheuthyroidism
GravesBasedow
diseaseNumber of
cases
n
54
25

%
65.06
30.12

The averageage group(years)medie ± DS
14 ± 2.4
14 ± 1.93

4

4.82

16 ± 0.5

Female

Male

n
47
20

%
87.03
80

n
7
5

%
12.97
20

4

100

-

-

Legend: ACT = chronic autoimmune thyroiditis

18 – 19 years
20 – 29 years
30 – 39 years
40 – 49 years
50 – 59 years
60 – 69 years
70 – 79 years

Number of
cases
n
%
11
1.7
29
4.46
48
7.38
168 25.84
219 33.7
118 18.15
57
8.77

Female
n
10
27
43
141
209
112
46

%
90.9
93.1
89.58
83.93
95.43
94.91
80.7

Male
n
1
2
5
27
10
6
11

%
9.1
6.9
10.42
16.07
4.57
5.09
19.3

Adult group was subdivided in function of the type at glycemic balance in 4 subgroups (Figure 1):
 group with type 1 diabetes represented by 60 cases(9.23%)
 group with type 2 diabetes accounted for 290 cases(44.61%)
 group with impaired glucose tolerance (IGT) accounted
for 183 cases (28.15%)
 group with fasting impaired glucose tolerance (IFG)
accounted for 117 cases (18%)

Case prevalence

50%Type of thy- Total
DM type 1 DM type 2 IGT
IFG
roid disease n
%
n %
n
%
n %
n %
Graves -Base- 147 22.61 6 10
60 20.69 43 23.49 38 32.47
Autonomous
thyroidadenoma

8

1.23

-

-

3

1.03

3

1.64

2

1.71

Amiodaroneinduced
hyperthyroidism

4

0.61

-

-

4

1.38

-

-

-

-

Differentiated thyroid
carcinoma(papillary,follicular)

21

3.23

3

5

10

3.49

2

1.09

6

5.12

Nodular goiter 11
with euthyroidism

1.7

-

-

7

2.41

2

1.09

2

1.71

1.66

125 43.1

Diffuse goiterwith euthyroidism

246 37.84 1

ACT (with goi- 208 32
ter, atrophic)

5

Sub acute
thyroiditis

0.77

81 44.26 39 33.33

50 83.33 77

26.55 52 28.41 29 24.78

-

1.38

-

4

-

-

1

0.85

Depending on “fasting” glucose, we assess the level ofglycemic balance in diabetic patients (Tables V and VI).
Table V. Glycemic balance in children and adolescents studied group
Very good

Good

Satisfactory

Unsatisfactory

44,61%

40%
28,15%

30%

n

%

n

%

n

%

n

%

26

31.32

12

14.45

9

10.84

36

43.37

18%

20%
10%

Table IV. The prevalence of thyroid disease in subjects with changes
in glycemic balance

dow disease

Table III. Distribution by age and sex of adult groupAge

the principal prevalence was with ACT (83.33%), followed by
Graves-Basedow disease (10%), thyroid carcinoma (5%) anddiffuse goiter with euthyroidism (1.66%).
In the group with type 2 diabetes, the main prevalence
was diffuse goiter with euthyroidism (43.1%), followed by ACT(26.55%) and Graves-Basedow disease (20.69%).
In the group with IGT, the main prevalence was diffusegoiter with euthyroidism (44.26%), followed by ACT (28.41%)
and Graves-Basedow disease (23.49%).
In IFG group the main prevalence was with diffuse goiter witheuthyroidism (33.33%), followed by Graves-Basedow disease(32.47%) and ACT (24.78%).

Table VI. Glycemic status in the studied group of adults

9,23%

Glycemicbalance0%
DM type 1

DM type 2

IGT

IFG

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

Regarding the type of thyroid disease, in the adult group
we obtained the results shown in Tab.IV. In type 1 diabetes caseDM type 1(n = 60)n%

DM type 2(n = 290)n%IGT(n = 183)n%IFG(n = 117)n%Very good8

13.33 151 52.06 151

82.52

78

66.66

Good

7

11.66

20.34 29

15.84

35

29.91

Satisfactory

10

16.66 25

8.62

1.64

4

3.42

Unsatisfactory

35

58.33 55

18.96 -

-

-

-

59

3

19

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The results of comparison between subgroups of lot of
adults are found in TableVII.
Table VII. Comparison (p) in blood glucose between sub lots of lot of adults
Glycemic balance

Very
good

Good

Satisfactory Unsatisfactory

DM type 1 vs. DM type 2< 0.001 0.1177

0.0586< 0.001

DM type 1 vs. IGT< 0.001 0.4289< 0.001< 0.001

DM type 1 vs. IFG< 0.001 0.0069

0.0019< 0.001

DM type 2 vs. IGT< 0.001 0.2208

0.0017< 0.001

DM type 2 vs. IFG

0.00720.0381

0.0648< 0.001

IGT vs. IFG

0.00160.00370.3193-

We observed highly significant differences (p<0.001) between the unsatisfactory glycemic balance who predominate incases with type 1 diabetes toward type 2 diabetes and otherchanges in glycemic balance and between very good glycemicbalance who predominate to other changes in glycemic balance
toward type 1 diabetes.
If the study group, Graves-Basedow disease was discovered in 4.82% cases in the group of children and in 22.61%
cases in the group of adults.
In the lot of children, in two cases the influence on the DM
was marked: one case was presented with advanced ketoacidosis (“fasting” glycemia 765 mg %) and 1 case in moderate
ketoacidosis (“fasting” glycemia 418 mg %). The rest had values
for blood glucose between 200-250 mg%. Under the treatment ofGraves-Basedow disease, there was an improvement in glycemicbalance with decreased initial insulin requirements.
In the case of type 1 diabetes in adults, in one case met
early ketoacidosis, in the remaining cases blood glucose values
being between 200-250 mg%. Under the treatment of thyroid
disease, glycemic balance was restored, with lower initial insulin
requirements.
In the case of type 2 diabetes in adults, only 4 cases had
glucose values between 200-250 mg%. Initially, they requirechanging the therapeutic regimen, with dose escalation oforal antidiabetic (OAD), the combination of two OAD, OAD
establishment in patients with diet and insulin treatment in 2
cases. After balance of Graves-Basedow disease, was restored
original scheme of treatment, and in those under insulin therapy
were low doses of insulin.
Changes in glycemic balance were diagnosed by OGTT
and required treatment with diet.
Between TSH and HbA1c value we found a very weakcorrelation (r = 0.22, p = 0.001) in all cases with diabetes andGraves-Basedow disease.
Correlation between serum TSH and HbA1c in all cases oftype 2 diabetes and Graves - Basedow disease was also very
weak (r = -0.10, p <0.001).
A number of studies show the influence of type of thyroid
disease on the metabolic balance. Thus, studies in Spain, Japan

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and the U.S. highlights the role of thyrotoxicosis in the development of diabetic ketoacidosis, it is meeting in these conditions
not only in patients with type 1 diabetes but also in those with
type 2 diabetes (4,13, 15, 17, 19).
Patients with Graves-Basedow disease should be closely
monitored both in terms of response to antithyroid therapy andin terms of glycemic balance.
Once stabilized disease, may decrease insulin requirements.
Also after stabilization of disease, blood glucose monitoring isnecessary to avoid hypoglycemia that can occur as a result of
lower insulin requirements.
In case of labile Graves-Basedow disease, which frequently
causing the glycemic decompensation, it is indicating surgery
treatment of the disease or therapy with I131.
Autonomous thyroid adenoma was found only at 8 casesin adults. We had not cases of autonomous thyroid adenoma andtype 1 diabetes, only with type 2 diabetes and other glycemicchanges. He manifested less severe thyrotoxicosis than GravesBasedow disease. In the group with type 2 diabetes, a single
case presented glucose values above 200 mg% All three cases
were balanced with insulin. Changes in glycemic balance werediagnosed by OGTT and required treatment with diet. All casesof thyroid adenoma were operated independently.
Differentiated thyroid carcinoma (papillary, follicular)
was discovered in 21 cases. All cases were operated, treatedwith I131, being then under suppression therapy with LT4. Because high doses of LT4, in one case of type 1 diabetes glucose
values were above 200 mg%, requiring a marked increase ininsulin doses. In other cases there was slight increase in insulindose, the dose of OAD and the establishment of the dietary inother glycemic changes. It is recommended that doses of LT4(suppressive therapy) remain unchanged, even worse glycemic
control, because if the dose of thyroid hormones decrease it is
the risk of developing systemic metastasis.
Amiodarone-induced hyperthyroidism was found only in
4 cases with type 2 diabetes. Poor glucose control was easily,with blood sugar below 200 mg%, 2 cases were treated with dietand 2 cases with OAD.
In the case of hypothyroidism, it causes impaired
carbohydrate metabolism, these changes were rarely clinicallysignificant. In hypothyroidism, the synthesis and release of
insulin is low. They cause a decrease in gluconeogenesis,
decreased glucose utilization in the periphery (14). Following is
an increased risk of hypoglycemia in individuals with diabetes(7). May decrease exogenous insulin requirements. Sometimes
produce dyslipidemia, including increased plasma triglycerides
and LDL cholesterol. Also, hypothyroidism may exacerbate
atherogenic manifestations and coexisting dyslipidemia in type2 diabetes. Treatment with thyroxin causes reversal of lipid
metabolism disorders
Chronic autoimmune thyroiditis (ACT) met in 65.06%
cases in children. In ACT with euthyroidism, without treatment,
glycemic balance has not changed. ACT with hypothyroidism
determinate an improvement in glycemic control with decreased
insulin requirements. The treatment with thyroid hormones in20

some cases led to increased insulin requirements, even obtaining
an unsatisfactory glycemic balance in 21 cases.
In the case of the group of adults ACT met in the 32% cases.
In ACT with euthyroidism, without treatment, glycemic balance
has not changed. ACT with hypothyroidism determinate an
improvement in glycemic control, with decreased insulin dose
and the necessary OAD. The treatment with thyroid hormone
determinate an unsatisfactory glycemic balance in 31 patientswith type 1 diabetes and in 11 cases with type 2 diabetes.
As a result, we increased dose of insulin and OAD. ACT withhypothyroidism (sub clinical and clinical) is frequently associatedwith dyslipidemia, leading to an increased risk of atherosclerosis
and cardiovascular disease.
Between the value of TSH and HbA1c we found a very
weak correlation (r <0.05, p <0.05) in all cases of diabetes andthe ACT.
The correlation coefficient between the serum TSH concentration and HbA1c in all cases with type 1 diabetes and ACT
was insignificant (r = -0.17, p = 0.40).
Also, the correlation coefficient between serum TSH andHbA1c in all cases with type 2 diabetes and ACT was insignificant(r = - 0.046, p = 0.30).
Dyslipidemia were seen in the group of children in five cases(four with hypercholesterolemia and 1 with hypertriglyceridemia),in the group of adults with type 1 diabetes in 25 cases (24with hypercholesterolemia and 1 with mixed dyslipidemia), in
the group of adults with type 2 diabetes in 62 cases (34 with
mixed dyslipidemia, 23 with hypercholesterolemia and 7 withhypertriglyceridemia), in the group of adults with IGT in 36
cases (17 with mixed dyslipidemia, 16 with hypercholesterolemiaand 3 with hypertriglyceridemia) and in the group of adults
with IFG in 24 cases (7 with mixed dyslipidemia and 17 withhypercholesterolemia).
Rotterdam study also showed that sub clinical hypothyroidism is a risk factor for cardiovascular disease (7). Other studies
have shown contrary results, sub clinical hypothyroidism is not
associated with an increased risk of death from cardiovasculardisease (2)Other authors have also shown the benefit of sub
clinical hypothyroidism treatment, besides achieving balance
thyroid function and decreased LDL-cholesterol (14).
Studies from Czech also show the influence of the association of various endocrine disorders on metabolic balance.
The most serious consequences are increased frequency of
hypoglycemia in hypothyroidism and occurrence of diabetic
ketoacidosis in case of thyrotoxicosis (20).
In hypothyroidism is an increased risk of mental retardationin children of hypothyroid mothers, and in pregnant women withdiabetes is frequently associated production of hypoglycemia byincreasing insulin secretion, decreased insulin requirements andby reducing liver glucose production (10).
Also, some authors suggest that the diagnosis of thyroid
diseases can be difficult. For example, poor glucose control
produces similar symptoms with hyperthyroidism as weight loss
despite increased appetite, marked weakness, fatigue, poliuro-

polydipsia syndrome.
Clinicians must be careful to not confuse hypothyroidismwith severe diabetic nephropathy: produce edema, fatigue, pallor
and weight gain.
In the study group, only in type 1 diabetes at two patients,
initial diagnosis was established Graves-Basedow disease,symptoms are weight loss despite increased appetite, marked
fatigue, asthenia. After, they were diagnosed through blood
glucose determination also with diabetes type 1.
A particular association is the association of type 1 diabetes
with autoimmune thyroid disease, particularly chronic autoimmune thyroiditis. In patients with ACT predominates hypothyroidism(clinical and sub clinical), while hyperthyroidism is rare (1).
American Thyroid Association (ATA) recommends testing
thyroid function in all patients over 35 years and thyroid function
revaluation every five years.
More commonly, testing is indicated in individuals at highrisk or those symptomatic. American Association of clinical
Endocrinology (AACE) recommends TSH screening in women
before pregnancy and during the first semester (19).
In 2005, AACE, ATA and Endocrinology Society (TES)
published a consensus about thyroid dysfunction (1). They
recommended determination of anti-TPO antibodies in patientswith sub clinical hypothyroidism because those with positive
antibodies have an increased risk of developing clinical thyroid
disease (8).
It is therefore recommended in patients with diabetes todetermine the TSH to assess thyroid function screening. In patients with type 1 diabetes must determine antiTPO antibodies
as predictors of autoimmune thyroid diseases.
Treatment is similar as in non-diabetic population. The
authors point out that inappropriate treatment with L-thyroxin may exacerbate angina pectoris and may increase
myocardial contractility and heart rate. Also recommend sub
clinical hypothyroidism treatment if patients have elevated
LDL-cholesterol exacerbated by hypothyroidism or antiTPO
antibodies detectable in serum. The authors concluded thatthyroid disorders are common in patients with diabetes andmay determinate metabolic disorders. It is therefore recommended regular screening of patients with diabetes, which
will allow early treatment. In patients with type 1 diabetes isrecommended determination of anti-TPO antibodies at diagnosis. In those with positive TPO antibodies is recommended
annual screening of TSH.
In patients with type 1 diabetes with antiTPO antibodies
negative TSH should be evaluated every 2-3 years. In patientswith type 2 diabetes should be evaluated TSH at diagnosis andthen at least at 5 years (11).
In the study group met severe ophthalmopathy in GravesBasedow disease in cohort of children in four cases, in adults with
type 1 diabetes in two cases, in patients with type 2 diabetes in
21 cases, in those with IGT in 15 cases and at those with IFG in
16 cases. A study in Greece shows in Graves-Basedow disease
that children are the same or even higher risk than adults todevelop infiltrative ophthalmopathy (12).
21

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CONCLUSIONS
If thyroid disease was first developing, excess of thyroid
hormone or administering thyroid hormone lied to an imbalanceof a pre-existing type 2 diabetes or unmasking latent one, andthe emergence of IGT and IFG.
Type 2 diabetes that appeared was easy, requiring only
diet therapy.
If the DM preceded thyroid disease with hyper function, its
appearance has led to imbalance of DM. Thus, cases of type 2
diabetes initially treated with diet requiring treatment with OAD
and those who received treatment with OAD required combination of OAD or even treatment with insulin.
In the case of type 1 diabetes, was a major imbalance,
manifested by diabetic ketoacidosis. Hyperthyroid syndrome
characteristic Graves-Basedow disease has led to a more severe
metabolic disturbances of diabetes mellitus type 1 compared tothat caused by chronic autoimmune thyroiditis and diffuse goiterwith euthyroidism. Thus, thyrotoxicosis has required increasing
doses of insulin, in parallel with the establishment of an energetic
antithyroid treatment.
In children and adolescents were observed thyrotoxicosis
resistant to usual therapy with antithyroid treatment, with incomplete and late onset of remission. Association between type 1
diabetes and Graves-Basedow disease requires systematic
dispensary of these patients due to the high risk of relapse
thyrotoxic syndrome.
Graves-Basedow disease and diabetes is a particularpathogenic entity, the presence of decompensate thyrotoxicosis
adversely affecting the evolution and prognosis of diabetes
mellitus. Because Graves-Basedow disease has a chronicevolution, being relatively difficult to stabilize even antithyroid
treatment, particularly its association with type 1 diabetes,
requires definitive correction of hyperthyroidism by surgicalthyroidectomy or radiation.
Dispensary of diabetic patients with associated thyroid
disease (including those with euthyroidism) covers annual
TSH monitoring for early detection of hypothyroidism. If thyroid
hypo function has no major implications for glycemic balance,
atherosclerotic complications of mixedema adversely affectlong-term prognosis of the association between diabetes mellitus
and hypothyroidism.
Therapeutic maneuvers required by the diagnosis of thyroid
cancer (total, treatment with I131 and suppressive therapy withhigh doses of thyroid hormones) can unbalance previouslycompensated diabetes. With damage to the metabolic balanceof diabetes, because high doses of thyroxin, it is recommended
higher doses of insulin, oral hypoglycemic agents respectively,
and not decrease the thyroxin dosage to avoid potential local
recurrence.
Diffuse and nodular goiter with euthyroidism required a
suppressive treatment with thyroxin up to the induction of sub
clinical hyperthyroidism. In case of a difficult control of diabetes
requires thyroidectomy.
In conclusion, the main changes that occur as a result ofinterrelation between diabetes - thyroid diseases are found in

Table VIII. Interrelation between DM-thyroid diseases (modif.17)
Clinical Condition

Effects on glycemicbalance

Effects on thyroid
function-

Decreased T3
Increased rT3
Decreased TSH
response to TRH
Decreased nocturnal peakof TSHDM in subjects with Worsening of glycemic
hyperthyroidism
control

Increasing severity of optic
neuropathy

Hyperthyroidism in Glucose intolerance inthe normoglycemic 50% of cases
subjects
DM in 2-3% cases-

Hyperthyroidismin subjects withdiabetes

Damage of diabetes
control-

Hypothyroidismin subjects withdiabetes

Predisposition to recur- rent hypoglycemia
Multiple
atherosclerotic
manifestations

Subjects with type1 autoimmune
diabetes-

DM at euthyroid
subjects

Increased prevalence of
thyroid diseases

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1. Balducci-Silano PL, Connor E, Maclaren NK. Association between
insulin dependent diabetes mellitus (Type 1) and other autoimmune
diseases. In: Diabetes Mellitus, a fundamental and Clinical Text. Le
Roith D., Taylor SI, Olefsky JM (eds), Lippincott-Raven, Philadelphia,
2002: 401 - 19
2. Cooper D. Subclinical hypothyroidism. N Engl J Med, 2001; 345:
260-265.
3. Dimitriadis G, Raptis SA. Thyroid hormone excess and glucose
intolerance. Exp.Clin.Endocrinol.Diabetes, 2001; 109: S 225-S 239.
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5. Greener M. Thyroid disease and diabetes. Clinical diabetes, 2000;
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6. Gunczler P, Lanes R, Soros A, Verdu L, Ramon Y, Guevara B, Beer N.
Coronary artery calcification, serum lipids, lipoproteins, and peripheral
inflammatory markers in adolescents and young adults with type 1
diabetes, J Pediatr, 2006; 149 (3): 320-3.
7. Hak AE, Pols HA, Visser TJ, Drexhage HA, Hofman A, Witteman CM.
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Ann Intern Med, 2000; 1232: 270-278.
8. Hossein GR, Tuttle M., Baskin HJ, Fish LH, Singer PA, McDermot
MT. Subclinical thyroid dysfunction: a joint statement on managementfrom the American Association of Clinical Endocrinologists, The American Thyroid Association, and the Endocrine Society. J Clin Endocrinol
Metab, 2005; 90: 581-585.
9. Johnson JL. Diabetes Control in Thyroid Disease, Diabetes Spectrum,

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Health and Human Development Diabetes in Early Pergnancy Study
Group: Declining insulin requirement in the late first trimester of diabetic
pregnancy. Diabetes Care, 2001; 24: 1130-36.
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Diabetes Care, 2003; 26 (1): 255-256.
12. Krassas GE, Gogarkos A. Thyroid-associated ophthalmopathy injuvenile Graves’disease – clinical, endocrine and therapeutic aspects,
J Pediatr Endocrinol Metab, 2006; 19 (10): 1193-206.
13. Kunishige M, Sekimoto E, Komatsu M, Bando Y, Uehara H, Iyumi
K. Thyrotoxicosis masked by diabetic ketoacidosis. Diabetes Care,
2001; 24: 171.
14. Mohn A, Di Michele S, Di Luzio R, Tumini S, Chiarelli F. The effectof subclinical hypothyroidism on metabolic control in children and ado-lescents with Type 1 diabetes mellitus, Diab. Med., 2002; 19; 70-3.
15. Saitoh T, Akamine N, Sakai T, Serizawa S, Tohno H, Horie T. Slowly
progressive insulin-dependent diabetes mellitus in an elderly patientwith Grave’disease, Intern Med, 2000; 39 (11): 943-6.
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INTERRELATIILE DINTRE DIABETUL ZAHARAT SI AFECTIUNILETIROIDIENEREZUMAT
Diabetul zaharat şi afecţiunile tiroidiene reprezintă două endocrinopatii frecvent întâlnite în populaţia generală. Deoarece insulina şi
hormonii tiroidieni sunt implicaţi în metabolismul celular, excesul sau deficitul unuia dintre aceştia determină tulburări funcţionale aleceluilalt. Scopul acestui studiu a fost de a determina influenţa afecţiunilor tiroidiene asupra diabetului zaharat, respectiv a diabetului
zaharat asupra afecţiunilor tiroidiene asociate. Lotul general studiat a fost reprezentat de 733 cazuri, cu vârste cuprinse între 7-79
ani.
Lotul studiat a fost subîmpărţit după criteriul vârstei în 2 loturi: lotul de copii şi lotul de adulţi. S-au folosit parametrii clinici, imagistici,
biochimici, hormonali, imunologici.
Cuvinte cheie: diabet zaharat, afecţiuni tiroidiene, interrelaţie

23

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THE EFFECT OF EPILEPSY ON THE AGE-RELATEDPROLONGATION OF THE P300 WAVE
MAJOR ZOLTÁN ZSIGMOND1, BUZOIANU ANCA DANA1, PERJU-DUMBRAVĂ LĂCRĂMIOARA2,MĂRGINEAN IOAN2, KRAUSZ LAJOS TIBOR1, VĂCĂRAŞ VITALIE2, BRUSTUREAN-BOTA
EMA2
1
Pharmacology Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
2
Neurology Department, „Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca

ABSTRACT
Background and purpose: Event related evoked potentials (ERPs), according to the literature, present disturbances in epilepsies. It
is known that P300 latency shows an age-related prolongation also. This study was designed to evaluate if the age-related prolongation of the latency of the auditory P300 is influenced by the presence of epilepsy.
Methods: 54 patients were included, and four groups were formed, a control group for the 20-40 age group, one for the 40-60 age
group, and two epilepsy groups divided using the same age criteria. Auditory P300 evaluation was performed.
Results: P300 latencies were significantly prolonged in the presence of epilepsy in both age groups, when compared with thematched controls, for all the investigated derivations. P300 latencies were significantly prolonged in the 40-60 group compared withthe 20-40 group for controls and epileptics as well. Concomitant presence of the two altering conditions, epilepsy and age, has no
augmenting effect, it presents only a linear additive effect on the prolongation.
Conclusion: The velocity of the age-related prolongation of the P300 latency is not influenced by the presence of epilepsy, underproper, but heterogenous treatment, meaning that the cognitive decline is linear with age, both in controls and epileptics.
Keywords: event-related evoked potential, P300, latency, age, epilepsy

INTRODUCTION
Evoked potentials, as EEG techniques, are recorded over
diverse cortical areas, generated as a response to external
stimuli, or as an image of a cognitive response induced by thesestimuli. Considering the parameters influencing the characteristics of the evoked potentials, these are classified in two major
categories: stimulus related potentials (SRPs) and event related
potentials (ERPs).
Among the studies investigating epilepsy, there are several
researches performed for the evaluation of ERPs, but there islittle consideration about the influence of the epilepsy on the age
related prolongation of P300.
The latency of the P300 component reflects the temporaldefinition of post-stimulus attentional processes (1). Cognitive
evoked potentials in epilepsies were stated to be a form of
measure for the associated cognitive deficits. Postictal changesof these potentials were reported in temporal lobe epilepsy (2),without interictal persistence (3). Increased latency of the P300
was reported also in children with occipital paroxysms (4). Other
studies performed on patients with idiopathic generalized epilepsy support the prolongation of the P300 latency in epilepsy(5). Convergent results were reported, showing only slight differences regarding the type of epilepsy (6,7), treatment options(8), seizure duration and frequency (9) and the morphology of

the P300 (10). The prolongation also differs according to thestimulus used to evoke the P300 wave, auditory P300 being
more sensitive, than the visual one (11). There are, on the other
hand, authors affirming that P300 latencies are not influenced
by the presence of epilepsy (12), but these opinions are stillin a minority. Others are underlining the importance of older
antiepileptic drugs, mainly valproic acid, as an important factor
contributing to the prolongation (13).
Even if there are so many different factors possibly contributing, there’s a need to further investigate the effect of epilepsy
and its treatment on congnition, mirrored by the parameters of
the cognitive evoked potential.
METHODS
The present study was performed, after informed consent
was obtained from the participants, on adults, between 20-60
years, admitted to the Neurology Clinic of Cluj-Napoca. The fourgroups were: group I of healthy controls, between 20-40 years(n=13), group II, patients with epilepsy between 20-40 years(n=17), group III of healthy controls between 40-60 years (n=10),
and group IV (n=15) of subjects between 40-60 with positive
diagnosis for epilepsy. Patients with cognitive deficits, diabetes
mellitus, dyselectrolytemias and psychiatric comorbidities were
excluded. The presence of any hearing disturbance was also

Received November 2010. Accepted December 2010. Adress for correspondence: Major Zoltan, „Iuliu Haţieganu” University of Medicine and Pharmacy, 400676 ClujNapoca, Negoiu no. 10/2 Street, Romania, phone: +40-740.075.796, e-mail: majorzsiga@yahoo.com

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an exclusion criterion.
The groups were formed, after applying the inclusion andexclusion criteria. Group I will be referred as Control 1 in the followings. Group II showed a positive diagnosis for epilepsy, withongoing treatment for it, and it was named Epi 1. Group III, named
Control 2, and was composed of healthy adults between 40-60,
and Group IV, Epi 2, epileptic patients from the same age group,
under antiepileptic treatment. P300 evaluation was performedon the selected patients. The aim of the study was to determine
the effect induced by epilepsy, as a symptom, or its treatment,on the velocity of the age-related prolongation of the P300 wave.
According to this, the epilepsy groups were heterogenous for thetype of epilepsy and for the applied treatment also.
A Medtronic Keypoint 4 device was used to record theP300 wave, stimuli being presented through earphones, according to the oddball paradigm, using a resolution of 100 ms,
50 μV per division, low pass frequency at 0.2 Hz, high passat 0.1 kHz, maximum tolerated impedance of the electrodes 1
kOhm, variable stimulation frequency, between 0.3 and 1 Hz,
percentage of the odd stimuli 15%, the frequency for odd stimuli
of 2000 Hz and the frequent ones of 1000 Hz. Recordings weremade with electrodes placed at Fz, Cz and Pz of the 10-20
system, reference electrode in A1 and A2, and the ground at
Fpz. Two evaluations were performed for each subject, with anumber of 150 averages, 22-23 odd sounds/evaluation. The
recorded parameter was the latency of the P300, mainly because this is the most robust measure that can be determinedwith this technique. The amplitude presents a high intra- andinterindividual variabity, this being the reason it was not taken
into account.
Statistical analysis was performed using SPSS version
number 17, after the calculation of means and standard errors (x ± s.e), implementing ANOVA and post-hoc Scheffè test, andafterwards two-way ANOVA, with a treshold for significance of
p<0.05.
RESULTS
The P300 wave was recorded in Fz, Cz and Pz of the 10-20
system. The latencies were compared at first between the threederivations, without significant differences (not shown).
Age becomes a grouping factor, by ordering patients according to it; then paired comparisons were made between Epi 1 andControl 1, and Epi 2 and Control 2, without significant differences(see below, Table I), so the control groups can be considered asage-matched. On the other hand, the distribution of the patientsusing as a criterion their sex is also matched between the agedefined groups (not shown).
The latencies of the P300 component presented a difference with a high global significance (ANOVA), when all
the groups were compared, and at the post-hoc evaluation(Scheffè) the latencies were found significantly prolonged foreach derivation on the epilepsy groups, versus the control
groups; the two control groups, and the two epilepsy groups
showed also significant differences at the paired analysis(Table I).

Table I. Means, standard errors, and significance thresholds, obtained fromthe statistical analysis performed on the groups
Age

Fz

Cz

Pz

x ± s.ex ± s.ex ± s.ex ± s.eEpi 1

27.38 ±
1.38

353 ± 8.23 357 ± 8.49

360 ±
8.38

Control 1

28.00 ±
1.51

326 ± 3.58 328 ± 3.56

330 ±
3.47

Epi 2

50.93 ±
1.93

389 ± 9.08 392 ± 8.80

395 ±
8.69

Control 2

50.33 ±
2.07

356 ± 6.01 358 ± 5.98

361 ±
5.96

ANOVA
Epi 1-Control 1-Epi
2-Control2

0.000

0.000

0.000

0.000

Epi 1 vsControl 1

0.976

0.048

0.033

0.027

Epi 1 vs
Epi 2

0.000

0.006

0.009

0.006

Control 1 vsControl 2

0.000

0.005

0.007

0.007

Epi 2 vsControl 2

0.887

0.020

0.019

0.012

ScheffèP

The second part of the study investigated if the presenceof epilepsy has an effect on the age related prolongation of
the P300 wave. We’ve used the two-way ANOVA test, anddetermined that even if both epilepsy and age, taken alone,
has a significant prolonging effect on the auditory P300 wave,
there is no significant effect of the interaction of the two factors(Tables II, III and IV).
Table II. The combined effect between age and epilepsy on the prolongationof the P300 wave registered in Fz: the interaction has no significant effect.
Dependent Variable: Fz

SourceType IIISum ofSquaresdf

Mean
SquareF

Sig.

PartialEta
SquaredCorrected
Model

.006a

3

.002

14.657.000.463Intercept

46.2851

46.285318736.764

.000

1.000

Epilepsy

.003

1

.003

20.409.000.286

Age_interval

.003

1

.003

20.937

.000

.291

1

1.410E-5

.097

0.757

.002

.000

Epilepsy *1.410E-5
Age_interval
Error

.007

51

Total

48.18655

Corrected
Total

.014

54

a. R Squared = .463 (Adjusted R Squared = .431)

25

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Table III. The combined effect between age and epilepsy on the prolongationof the P300 wave registered in Cz: the interaction has no significant effect.
Dependent Variable: Cz
SourceType IIISum of df
Squares

Mean
SquareF

Sig.

PartialEta
SquaredCorrected
Model

.006a

3

.002

14.644.000.463Intercept

46.424146.424322267.232

.000

1.000

Epilepsy

.003

1

.003

21.496.000.297

Age_interval

.003

1

.003

19.944.000.281

Epilepsy *Age_interval

7.631E-6

1

7.631E-6

.053

0.819

.001

Error

.007

51

.000

Total

48.33455

Corrected
Total

.014

54

a. R Squared = .463 (Adjusted R Squared = .431)
Table IV. The combined effect between age and epilepsy on the prolongationof the P300 wave registered in Pz: the interaction has no significant effectDependent Variable: Pz
SourceType IIISum ofSquaresdf

Mean
SquareF

Sig.

PartialEta
SquaredCorrected
Model

.006a

3

.002

15.756.000.481Intercept

46.556

1

46.556339028.480

.000

1.000

Epilepsy

.003

1

.003

23.384.000.314

Age_interval

.003

1

.003

21.160.000.293

Epilepsy *Age_interval

1.110E-5

11.110E-5

Error

.007

51

.000

Total

48.47355

Corrected
Total

.013

54

.081

0.777

.002

a. R Squared = .481 (Adjusted R Squared = .450)

DISCUSSION
For the beginning, the possibility that the obtained results
could be more significantly validated in case of higher numberof subjects should be emphasized.
The P300 wave appeared with a prolongation of the latency
in the presence of epilepsy, regardless the investigated age
interval, 20-40, respectively 40-60. The prolongation is in accordance with most of the studies found in the recent literature.
This increase of the latency should be cautiously interpreted
when the source of the prolongation is investigated, because it
can be influenced not only by the presence of epilepsy, but also
by the length of the exposure to antiepileptic treatment, and thetype of this treatment.
As it was already stated, the P300 wave shows an approximate prolongation rate with age, of 1.6 ms/year (14). Our
study concludes that this rate is not influenced by the presence

CONCLUSIONSThe presence of epilepsy, in both investigated age groups,
causes a highly significant prolongation of the P300 wave,
possibly related to either a cognitive deficit, or at least partly aconsequence of the ongoing treatment. The age related prolongation’s velocity is not increased by the presence of epilepsy,
maintaining its linearity regardless of the length of the diseaseor the length of exposure to antiepileptic drugs.
REFERENCES
1. Kutas M, McCarthy G, Donchin E. Augmenting mental chronometry:the P300 as a measure of stimulus evaluation time. Science, 1977;
197(4305): 792-5.
2. Abubakr A, Wambacq I. The localizing value of auditory event-related
potentials (P300) in patients with medically intractable temporal lobe
epilepsy. Epilepsy Behav., 2003 Dec;4(6): 692-701.
3. Bocquillon P, Dujardin K, Betrouni N, Phalempin V, Houdayer E,
Bourriez JL et al. Attention impairment in temporal lobe epilepsy: A
neurophysiological approach via analysis of the P300 wave. Hum Brain
Mapp., 2009;30(7): 2267-77.
4. Gokcay A, Celebisoy N, Gokcay F, Atac C. Cognitive functions
evaluated by P300 and visual and auditory number assays in children
with childhood epilepsy with occipital paroxysms (CEOP). Seizure,
2006;15(1):22-7. Epub 2005 Dec 15.
5. Sunaga Y, Hikima A, Otsuka T, Nagashima K, Kuroume T. P300
event-related potentials in epileptic children. Clin Electroencephalogr.,
1994;25(1):13-7.
6. Soysal A, Atakli D, Atay T, Altintas H, Baybas S, ArpacI B. Auditory
event-related potentials (P300) in partial and generalized epileptic
patients. Seizure, 1999; 8: 107-110.
7. Celebisoy N, Kisabay A, Gökçay F, Gökçay A. Evaluating cognitive
functions with visual and auditory number assays and P300 in children
with epilepsy. Brain Dev., 2005; 27(4): 253-8.
8. Ozmenek OA, Nazliel B, Leventoğlu A, Bilir E. The role of event related
potentials in evaluation of subclinical cognitive dysfunction in epileptic
patients. Acta Neurol Belg., 2008; 108(2): 58-63.
9. Caravaglios G, Natalè E, Ferraro G, Fierro B, Raspanti G, Daniele

Fiziologia - Physiology z 2010.20.4 (68)

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of epilepsy, although the overall values in every time point are
higher for epileptics, than for normal controls. This is the reason
we think epilepsy, or it’s treatment, or both, have a static roleon the velocity of the age-related prolongation of P300, whenepilepsy is controlled, maintaining the linearity of the effectinduced by age.
While in other diseases placebo is used in situations requiring discrimination between different variable effects, epilepsy
has the property of causing potentially life-threatening states
if left untreated, so it is an ethical issue to be solved in orderto define the primary role of either the epilepsy or its treatment
is causing the investigated effect on the evoked potential. Alsoin such situation we might be able to have healthy controls
under antiepileptic treatment, which is also not acceptable forsuch study conditions. If epilepsy alone has the effect, than it
should be emphasized that a proper treatment is needed for abetter control, less seizures meaning better long-term cognitive
performances; if treatment contributes, than newer agents, withless side effects, should be recommended.

26

O. Auditory event-related potentials (P300) in epileptic patients. Neurophysiol Clin., 2001; 31(2): 121-9.
10. Wambacq I, Abubakr A. Auditory event-related potentials (P300)
in the identification of psychogenic nonepileptic seizures. Epilepsy
Behav., 2004; 5(4): 503-8.
11. Duncan CC, Mirsky AF, Lovelace CT, Theodore WH. Assessment of
the Attention Impairment in Absence Epilepsy: Comparison of Visual and
Auditory P300. Int J Psychophysiol., 2009 August; 73(2): 118-122.
12. Sun W, Wang W, Wu X, Wang Y. Antiepileptic drugs and the sig-

nificance of event-related potentials. J Clin Neurophysiol., 2007 Jun;
24(3): 271-6.
13. Panagopoulos GR, Thomaides T, Tagaris G, Karageorgiou CL.
Auditory event related potentials in patients with epilepsy on sodium
valproate monotherapy. Acta Neurol Scand., 1997 Jul; 96(1): 62-4.
14. Goodin D, Desmedt J, Maurer K, Nuwer MR. IFCN recommended
standards for long-latency auditory event-related potentials. Report ofan IFCN committee. Electroencephalography and clinical Neurophysiology, 1994; 91: 18-20.

THE EFFECT OF EPILEPSY ON THE AGE-RELATED PROLONGATION OF
THE P300 WAVEREZUMAT
Introducere si scop: Conform datelor din literatura, potentialele evocate relationate evenimentului (ERP) prezinta anumite neregularitati in epilepsie. Este cunoscut faptul ca latenta P300 prezinta, de asemenea, alungire relationata varstei. Acest studiu evalueaza
influenta prezentei epilepsiei asupra alungirii latentei P300 relationata varstei.
Metode: Au fost inclusi in acest studiu 54 de pacienti si au fost formate 4 grupuri de studiu, un grup de control cu varsta cuprinsa
intre 20 si 40 ani, un alt grup control cu varsta intre 40 si 60 ani si doua grupuri de pacienti cu epilepsie, stabilite conform acelorasi
criteria de varsta. S-a efectuat evaluarea auditiva a P300.
Rezultate: Latentele P300 au fost semnificativ prelungite in prezenta epilepsiei pentru ambele grupuri de varsta, comparativ cu
grupurile de control corespunzatoare, in toate derivatiile investigate. Latentele P300 au fost semnificativ prelungite in grupul devarsta 40-60 ani, comparativ cu grupul de varsta 20-40 ani, atat pentru control, cat si pentru grupul cu epilepsie. Prezenta simultana
a celor doua conditii determinante, epilepsie si varsta inaintata, nu a avut un efect augmentativ, prezentand doar un efect aditiv liniar
asupra prelungirii.
Concluzie: Viteza de alungire a latentei P300 relationata varstei nu este influentata de prezenta epilepsiei, in cadrul unui tratament adecvat, heterogen, ceea ce demonstraza ca declinul cognitiv este linear cu varsta, atat la grupul de control, cat si la cel cu
epilepsie.
Cuvinte cheie: potential evocat relationat evenimentului, P300, latenta, varsta, epilepsie

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OXIDANTS-ANTIOXIDANTS BALANCE IN ORAL LICHENPLANUSIOANA SCROBOTA1, TEODORA MOCAN2, ADRIANA FILIP2, DOINA DAICOVICIU2, GRIGOREBACIUT3
1
CMI Dr. Ioana Scrobota, Oradea, Bihor, Romania
2
Department of Physiology, “Iuliu Hatieganu” University of Medicine and Pharmacy Cluj Napoca, Romania
3
Department of Oral, Craniomaxillary and Cervicofacial Surgery, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj Napoca, Romania

ABSTRACT
Lichen planus is a chronic inflammation of unknown etiology. The disease has been most often reported to affect the oral mucosa.
The most dangerous complication of oral lichen planus (OLP) is oral squamous cell carcinoma. Recently, oxidative stress has been
implicated in the pathogenesis of OLP. The aim of this study is to evaluate the status of oxidative stress and the antioxidant defense
system in patients with OLP. We evaluated and compared the levels of a series of oxidative stress markers in patients with OLP withthat of normal controls. Serum levels of malondialdehyde, protein carbonyl, sulphydryl groups, hydrogen donor and glutathione were
measured. Malondialdehyde (MDA), carbonyl protein (PC) and glutathione were increased and sulphydryl groups (SH) and hydrogen
donor (HD) were decreased in patients compared to controls. In conclusion our results point to a disturbance in the prooxidantsantioxidants balance in OLP and demonstrate the involvement of oxidative stress in the pathogenesis of this disease.
Key words: prooxidants-antioxidants balance, oxidative stress, oral lichen planus

INTRODUCTION
Lichen planus is a chronic inflammation of unknown etiology. It involves the skin, mucous membranes, genitalia, nails,
and scalp. The prevalence of LP is 1-2% and it is seen mostfrequently in middle aged patients more commonly in womenthan in men (31). The disease has been most often reported toaffect the oral mucosa (29).
Although the exact etiology is unknown OLP is recognized
as an autoimmune disease in which activated T cell releasecytokines leading to the attraction of inflammatory cells andthe destruction of keratinocytes by cell mediated cytotoxicity(9,21). Recently oxidative stress has been implicated in thepathogenesis of OLP (3).
The probability of malignant transformation is low still themost dangerous complication of OLP is oral squamous cell carcinoma (OSCC) (22,24). The accumulation of 8-nitroguanine and8-oxoG in the tissue of patients with OLP and OSCC (6) leaded
to the conclusion that cell mediated DNA alteration via oxidative
stress may play a key role in carcinogenesis of OLP (16-7).
The aim of this study is to evaluate the status of oxidative
stress and the antioxidant defence system in patients withOLP.
MATERIALS AND METHODSThis study included 9 patients with OLP and 4 healthy volunteers. All subjects were retrieved from The Department of Oral,
Craniomaxillary and Cervicofacial Surgery of “Iuliu Hatieganu”
University of Medicine and Pharmacy, Cluj Napoca, Romania.
This work has been approved by the ethical committee of “Iuliu
Hatieganu” University of Medicine and Pharmacy and informed

consent was taken from all the participants in this study. All
patients were subjected to a thorough history regarding dietary
habits and addiction and clinical examination.
Blood samples (5ml) were obtain from patients and controls
by venous arm puncture and were centrifugated for 5 minutes
at 3500 rpm. After the plasma was obtained it was immediately
frozen and kept at a temperature of -800 C until it was processed.
Serum MDA was established by fluorescein dosage, according
to Conti (7). The concentration values were expressed in nmol/ml. PC determination was based on Reznick method (26). The
results were expressed in nmol/mg protein. The assessment ofability of hydrogen donor was measured by Janaszewska method
and expressed in inhibition% (14). Total SH groups of serum wereassayed according to the method of Hu (13). The concentration
was expressed in micromol/ml. The serum GSH level was examined using Ellman method and expressed in nmol/ml.
Statistical analysis
Data normality was assessed using Kolmogorov-Smirnov
test. Data were analyzed using the a nonparametric KruskallWallis test for overall groups comparison, Mann-Whitney U Test/
Student for the comparison of two groups. The correlations between the qualitative variables were analyzed by Chi square test.
Adjustments were used using stratification of groups to exclude
the effect of possible error factors. SPSS 17.0 (Chicago, Il,USA)
statistical package was used to analyze all data.
RESULTS
General characteristics and correlations
The average age of the participants was 47.27 ± 17.83 years

Received August 2009. Accepted October 2009. Address for correspondence: Delia Cheptanariu, MD, Research Centre in Preventive Medicine, V. Babes no 16,Timişoara 300226, phone/fax. 0256-490412, e-mail: dorodel@yahoo.com

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old, significantly higher in patient’s group (p = 0.007) (Table 1).
Subjects in the control group have reported to consume one toxic
substance (alcohol/nicotine/caffeine) while subjects in the tested
group consume 1-3 types of drugs. The correlation between theuse of drugs and the group origin was not statistically significant(p = 0.246) (Table I). White reticular pattern of OLP was foundin all the subjects in the test group (Figure 1).

Serum
Median MDA levels were increased in the tested group
3.0 (1.10-6.70) versus control group 1.8 (1.2-2.10). The simple
comparison between groups has given values of 0.150 (notsignificant). However, after the age adjustment, the difference
was highly significant (p < 0.0001) (Figure 2).

Fig. 2. Serum MDA levels in tested group and controls

Similar results were obtained for the PC determination. Statistical data showed a value of 1.7 (1.0-3.2) in the patient’s groupand 1.2 (1.0-1.6) in the healthy group. Non-adjusted significancewas p = 0.089 (marginally significant). After age adjustment, thedifference was highly significant p < 0.0001 (Figure 3).

Fig. 3. Serum PC levels in tested group and controls

HD median level was decreased in the tested group 47.7
(38.5-58.2) as compared to control group 53.2 (47.7-58.3). The
difference was not statistically significant (p = 0.133). However,after age adjustment it became highly significant (Figure 4).
Fig.1. Clinical aspect of oral lichen planus
Table I. Characteristics of subjects
Variable

Controls

Tested groupAge (median ± SD)

28.25±2.9854.18±15.67

Toxic substances, no. (%)
Absent0(0%)

2(16.7%)

1 substance4(100%)

5(41.7%)

2 substances0(0%)

1(8.3%)

3 substances0(0%)

4(33.3%)Fig. 4. Serum HD levels in tested group and controls

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Median serum SH was lower in patients with OLP - 0.32
(0.15-0.54) - then in healthy participants - 0.39 (0.37-0.51). The
unadjusted level of significance was p = 0.026, while after adjustment, the alpha level was p < 0.0001 (Figure 5).

Fig. 5. Serum SH levels in tested group and controls

The median level of glutathione (GSH) was higher in patient’s serum as compared to control group - 7.12 (3.40-11.90)
versus 4.17 (3.05-6.60). Statistical parameters, such as p value,
were different in the absence or after age adjustment - p = 0.090
and p < 0.0001, respectively (Figure 6).

Fig. 6. Serum glutathione levels in tested group and controls

DISCUSSION
Oxidative stress is a disturbance of the prooxidantsantioxidants balance in favor of the prooxidants (8,12,30). This
usually results in the production of free radicals that can damage
cell membranes through lipid peroxidation, as well as numerous
cellular molecules such as proteins, nucleic acids, amino acids,
carbohydrates, and vitamins (2,23,28).
MDA, the final product of lipid peroxidation is considered agood biomarker of oxidative stress. We found an increased serum
level of MDA in patients versus healthy subjects. Our results
indicate the implication of oxidative stress in the pathogenesisof OLP. These findings are in corcondance with several studies
that demonstrated a significant increase in lipid peroxidation
product MDA, increased oxidative DNA damage, increased
protein oxidation, and disturbed enzymatic antioxidant defenses
in lichen sclerosis vulvae tissue specimens. In addition anotherstudy reported higher serum levels of MDA in LP patient groupthan the control group and high MDA levels in LP, leukoplakia,

CONCLUSIONSIn conclusion in patients with oral lichen planus there isevidence of the disturbance in the pro-oxidants-antioxidantsbalance characterized by high levels of pro-oxidants and decrease in the protective capacity ensured by antioxidants which
proves the implication of oxidative stress in the pathogenesisof this disorder.
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lipid peroxidation in human subjects with oral lichen planus. Int J Dent
Hygiene, 2009;7(4): 246-250.
2. Aly DG, Shahin RS. Oxidative stress in lichen planus. Acta Dermatoven APA, 2010;19(1): 3-11.
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Fiziologia - Physiology z 2010.20.4 (68)

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and cancer (1).
Generation of carbonyl groups on amino acid side chainsis one of the most commonly studied and used markers forprotein oxidation (20). In our study the values of PC in patient’s
serum were higher than in controls. This point out to the involvement of oxidative stress in the pathogenesis of the disease
we studied. Other works showed increased protein oxidation,
DNA damage, lipid peroxidation product MDA, and disturbed
enzymatic antioxidant defenses in lichen sclerosis vulvae tissue
specimens (27).
Serum hydrogen donor capacity (HD) is an expressionof the total antioxidant capacity of the serum because it
is evidencing the amount of reducing substance that has
neutralized ROS. The level of DH was decreased in the seraof our patients compared with normal individuals. We think that
this reduction is due to the decrease in the protective capacity
ensured by antioxidants. Increased oxidative stress and lower
antioxidant capacity was found in lichen sclerosis vulvae tissue
specimens (27), in serum of patients with OLP and in OSCC
saliva, sera and tissue samples (4).
SH groups are very susceptible to oxidation. When theorganism is exposed to oxidative stress they are the firstantioxidants to be sacrificed (10,11). In our study the level of–SH was lower in the test group than in controls. This decrease
illustrates the intensity of the oxidative stress in OLP. The results
are in concordance with other studies that have recorded lower
levels of serum –SH and higher levels of serum MDA in patientswith OLP versus healthy controls (33).
Glutathione is the most important non-enzymatic
antioxidant. GSH can act as a nucleophile substrate as well
as a reducing agent interacting with various free radicals (19. It
plays an important role in detoxification process (32). We foundhigher levels of serum GSH in tested group compared with
controls. Raised serum levels of GSH has also been reported
in OSF, leukoplakia, and oral cancer (5,15,18,25). We think thatprobably this is due to increased resistance to the cytotoxic
effects of xenobiotics of altered cells who can selectively growin a toxic environment relative to the normal cells.

30

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6. Chaiyarit P, Ma N, Hiraku Y et al. Nitrative and oxidative DNA damage
in oral lichen planus in relation to human oral carcinogenesis. Cancer
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7. Conti M, Morand PC, Levillain P et al. Improved Fluorometric Determination of Malonaldehyde. Clin Chem, 1991; 37(7): 1273-1275.
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13. Hu ML. Measurement of protein thiol groups and gluthatione inplasma. Methods Enzymol, 1994;233: 380-5.
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and ceruloplasmin and red cell glutathione and antioxidant enzymes inpatients of oral cancer. Indian J Cancer, 1986;23: 36-42.

19. Meister A. Glutathione metabolism and its selective modification. J
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2000;17(8):687–693.
21. Middel P, Lippert U, Hummel KM, et al. Expression of lymphotoxinalpha by keratinocytes: a further mediator for the lichenoid reaction.
Pathobiology, 2000;68(6): 291-300.
22. Mignogna MD, Fedele S, Lo Russo L et al. Immune activation andchronic inflammation as the cause of malignancy in oral lichen planus:is there any evidence? Oral Oncol, 2004;40(2):120-130.
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24. Rajentheran R, McLean NR, Kelly CG et al. Malignant transformationof oral lichen planus. Eur J Surg Oncol, 1999;25(5): 520-523.
25. Rao A, Rao N, Bajaj P, Renjith G, Kondapi K, Suneetha N. Levels of
glutathione, ceruloplasmin and malondialdehyde in oral leukoplakia andoral squamous cell carcinoma. Malays Dent J, 2002; 23: 85-90.
26. Reznick AZ, Packer L. Oxidative damage to proteins: Spectrophotometric method for carbonyl assay. Methods Enzymol, 1994; 233:
347-357.
27. Sander C, Cooper S, Ali I et al. Decreased antioxidant enzyme
expression and increased oxidative damage in erosive lichen planusof the vulva. BJOG, 2005;112(11): 1572-75.
28. Schafer FQ, Buettner GR. Redox environment of the cell as viewed
through the redox state of the glutathione ulfide/glutathione couple. Free
Radic Biol Med., 2001;30(11):1191-212.
29. Scully C, Beyli M, Ferreiro MC et al. Update On Oral Lichen Planus.
Etiopathogenesis and Management. Crit Rev in Oral Biol & Med, 1998;
9:86-122.
30. Sies H. Oxidative stress: introductory remark. In H. Sies(Ed): Oxidative Stress. London: Academic Press 1985, 1–7.
31. Sugerman PB, Savage NW, Walsh LJ et al. Disease mechanisms
in oral lichen planus. a possible role for autoimmunity. Australas J
Dermatol 1993;34(2):63–9.
32. Tache S. Oxidanţii şi antioxidanţii. În: A. Mureşan, în: A. Mureşan,S. Tache, R. Orăsan (sub red.), Stresul oxidativ în procese fiziologiceşi patologice, ed. Todesco, Cluj-Napoca, 2006, 1-27.
33. Upadhyay RB, Carnelio S, Shenoy RP et al. Oxidative stress andantioxidant defense in oral lichen planus and oral lichenoid reaction.
Scand J Clin Lab Invest 2010;70(4):225-228.

BALANTA OXIDANTI-ANTIOXIDANTI IN LICHENUL PLAN ORALREZUMAT

Lichenul plan este o inflamatie cronica de etiologie necunoscuta. Afectiunea intereseaza mai frecvent mucoasa orala. Cea mai periculoasa complicatie a lichenului plan oral (LPO) este carcinomul spinocelular oral. Recent stresul oxidativ a fost implicat in patogeneza
LPO. Scopul aceste lucrari este de evaluare a stresului oxidativ si a apararii antioxidante la pacientii cu LPO. Am determinat si amcomparat nivelul mai multor markari ai stresului oxidativ la pacientii cu LPO fata de martori sanatosi. Au fost dozate nivelele serice
ale malondialdehidei, proteinelor carbonilate, gruparilor sulfhidril libere, donorilor de hydrogen si a glutationului. Valorile malondialdehidei (MDA), a proteinele carbonilate (PC) si glutationului au fost crescute in timp ce gruparile sulfhidril libere (-SH) si donorii de
hIdrogen (DH) au fost scazute la lotul testat comparativ cu lotul martor. In concluzie rezultatele noastre indica o dereglare a balanteioxidanti-antioxidanti in LPO si demonstreaza implicarea stresului oxidativ in patogeneza acestei afectiuni.
Cuvinte cheie: balanta oxidanti-antioxidanti, stres oxidativ, lichen plan oral

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EXHALED NO AND RISK FOR ASTHMA IN PATIENTS WITH
ALLERGIC RHINITISIOANA ADRIANA BUJOR, IOANA CORINA BOCŞAN, DIANA DELEANU, VICTOR CRISTEAUniversity of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj Napoca, Romania

ABSTRACT
Background: The relationship between the upper and lower airways is well known. Allergic rhinitis is considered a risk factor inasthma development, and is also co-morbidity in asthma. Family history of asthma is known as a risk factor in asthma developing.
The aim of the study was aimed to evaluate the risk of asthma in patients with allergic rhinitis using exhaled nitric oxide (FENO).
Methods: 34 pts with allergic rhinitis were evaluated with Nioxmino for measuring FENO. The medical history of each patient andalso the five nasal symptoms score were evaluated. Skin pick test were performed for inhaled allergens (house dust mites, pollens,cat dander, dog dander, moulds).
Results: 15 pts (44.11%) were women (sex ratio f:m = 1:1.26). Mean age of the pts is 28.77 ± 10.38 years. All patients were allergic,
52.9% polysensitized more than one allergen. It was found a significant statistical correlation between family history of asthma an
increased values of FENO (p = 0.001). But between polysensitized patients and monosensitized patient there was no correlation
with FENO (p = 0.19), and also no correlation of FENO and rhinitis symptoms score (p = 0.08).
Conclusion: Increased values of FENO could be evaluated as a risk factor for developing asthma in patients with allergic rhinitis.
In allergic rhinitis severities of symptoms score was not a predictive factor in developing asthma. There was no correlation between
FENO and polysensitization.
Key words: exhaled nitric oxide, asthma, rhinitis, family history

INTRODUCTION
During the atopic march the evolution from allergic
rhinitis to asthma was evidenced. This two diseases co-exists often, could be named “one allergic disease” (1,2). Allergic rhinitis is
the most common IgE mediated disease and is highly increased
as prevalence during the last years (3). Allergic rhinitis is a risk
factor for asthma (4), and can appear before or after asthma.
Allergic inflammation is the key to understand this disease, andalso the evolution to co-morbidities, especially asthma.
Allergen inhalation leads to mast cell degranulation innasal mucosa, and release of mediators, mainly histamine andleukotriens. Th2 cytokines are responsible for the recruitment
of inflammatory cells in the tissues, including eosinophils, neutrophils and Th2 lymphocytes (5). The link between the upperand lower airways is not completely understood, it is possible
to explain this concept by the fact that inflammatory cells andmediators from nasal secretions enter in the lower airways
through inhalation and aspiration, and here they can act as a
trigger factor in lower airways inflammation (6).
The question “Which patients with allergic rhinitis will develop
asthma?” in the “united airways concept” is important for every
practitioner when we have a patient with allergic rhinitis in frontof us.
There is a hypothesis that eosinophils recruited in allergic
inflammation via expression of adhesion molecules (7), may
induce NO-syntetase in bronchial epithelial cells. Exhaled nitricoxide (FENO) is known as a marker of airways eosinophilic
inflammation in lower airways (8). Several studies showed thatpatients with atopic asthma have increased values of FENO

than non-atopic asthma. IgE- mediated inflammation leads toelevated values of FENO. Also studies have demonstrated thatpatients with hay fever during pollen season have an increased
values on FENO, even if they had not or had mild asthma
symptoms (9).Other study demonstrate that in patients withallergic rhinitis there are increased values of exhaled NO andadenosine compared with healthy subjects, those data suggeststhat in allergic rhinitis there is an subclinical inflammation in thelower airways (10). In allergic rhinitis an increased in FENO onallergen exposure, particularly in hyperresponsive subjects may
be suggestive of airway inflammation and an increased risk fordeveloping asthma (11,12). FENO measurements using Nioxmino are less expensive than other measurements for asthma
and very easy to use (13).
Genetic studies in allergies shown that there is a number ofsusceptibility genes could contribute to the allergic process, andis possible that the “allergic disease genes” and the environment
lead to a phenotype which can be asthma and/or allergic rhinitis
and/or atopic dermatitis (14).
Clinical aspect of asthma can vary a lot from classicalbronchospasm to chronic cough or only effort dyspnoea (2).
There are patients with allergic rhinitis with rare and mild asthma
symptoms which are not remark due to a severe rhinitis. The
difference between rihinitis patients with or without asthma symptoms seems to be a question of perception of dyspnoea. FENO
measurements indicate that dyspnoea could be associated withincreased inflammation in peripheral airways (15).
The aim of this study was to investigate the risk of asthma inpatients with allergic rhinitis using exhaled nitric oxide (FENO).

Received October 2010. Accepted November 2010. Address for correspondence: Adriana Bujor, MD, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj
Napoca, Romania, e-mail: adriana_bujor@yahoo.com

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MATERIALS AND METHODS
Subjects
A total of 34 patients with allergic rhinitis were evaluated with
Nioxmino® for measuring FENO. Doctor’s asthma diagnoses
according to Global Initiative for Asthma (GINA) in any patient
evaluated was not an inclusion criteria. Smokers and patientswith respiratory infections within 4 weeks were not included in
the study. Positive skin prick tests were mandatory for inclusion.
An accurate family history also was obtaining from patients andtheirs family. Patients who cannot provide an accurate family
history of diseases were excluded. Each patient had to complete
the five nasal symptoms score before any measurements.
All patients were attending in Allergy Department of “Octavian Fodor” Emergency Hospital from Cluj Napoca, and they
have given the inform consent to participate in the study.
None of the patients use medication 2 weeks before
evaluation. Were excluded patient that could not perform FENO
measurements
Methods
Allergy screening was made by skin prick tests (Hal Allergy, The
Netherlands) (16). A panel of common inhaled allergens was usedwhich included: house dust mites (Dermatophagoides pteronyssinus, Dermatophagoides farinae), grasses pollens mix, mug worth,birch, cat dander, dog dander, mould (Alternaria alternata).
Five nasal symptoms score
All patients participating in the study complete the five nasal
symptoms score. Rhinorrhea, nose itching, nasal stuffiness,sneeze, eyes itching were evaluated retrospectively in 12 hours.
They had to interpret the symptoms on a scale from 0 to 3 (0-absence, 1-mild, 2-moderate and 3-severe). The classification ofseverity score was according to ARIA guide1. Symptoms score over
6 included the patient as moderate-severe allergic rhinitis.
Fractional exhaled nitric oxide
All measurements were performed in accordance to recommendations of American Toracic Society, with Nioxmino®
(Aerocrine, Sweden) and defined in parts per billion (p.p.b.). No
maneuver of spirometry was taken before the test, and all subjects were in seated position when measurements were made.
Participants inhaled NO-free air through the mouth piece to totallung capacity, and the exhaled constantly (flow rate was assure
as 50ml/min) with a visual feedback display. For patients with pollen allergy measurements were made during pollen season.
Statistical analysis
It was a small group of patients in which we used Microsoft
Excel for dates, and Student Test for obtaining p value. A p value
under 0.01 was considered significant.
RESULTS
Patient’s characteristics
Thirty-four patients with allergic rhinitis were investigated.

15 pts (44.11%) were women (sex ratio f : m=1:1.26). The age
range from 11 to 48 years, patients mean age were 28.77±10.38
years. All patients have skin prick test positive to at least one
allergen, a positive skin prick test was considered at least 3 mmdiameter with histamine at least 5 mm diameter. Of those 52.9%
were polisensitizied to more than one allergen (Table I).
Table I. Sensitization at skin prick tests

Allergen

House
dust
mites

PollensCat
dander

Mould

Positive SPT
per allergen

25

24

4

3

FENO values
Between polysensitizied patients and monosensitizied
patient there was no statistical correlation with FENO values,
p = 0.19 (Figure 1).
9

0

8

0

7

0

6

0

5

0

4

0

3

0

2

0

1

0

41.21±36.99

32.13±19.48
p=0,190

Monosensitization

Polisensitization

Monosensitization Polisensitization

Fig. 1. FENO values correlation with sensitization

As severity of rhinitis patients who have five nasal symptoms score over 6 were included as moderate severe allergic
rhinitis patients. Medium FENO values were higher in moderate
severe allergic rhinitis group (42.33 ± 35.29 vs. 28.27 ± 18.49),
but results showed a non significant statistical correlation, p =0.08 (Figure 2).
9

0

8

0

7

0

6

0

5

0

4

0

3

0

2

0

1

0

42.33±35.29

28.27±18.49
p=0.080

MildPAR

Moderate/Severe PAR

MildPAR Moderate/Severe PAR

Fig. 2. FENO values correlation with severity of allergic rhinitis

Genetic predisposition for asthma was evaluated with an
33

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accurate family history of the patients. Patients with family historyof asthma had higher FENO values than patients with no asthma
history (49.44 ± 32.91 vs.22.67 ± 18.95), and the results were
statistical significant with p = 0.002 value (Figure 3).
9

0

8

0

7

0

6

0

5

0

4

0

3

0

2

0

1

49.44±32.91

REFERENCES
p=0.00222.67±18.95

00Family Hystory of Asthma

No Family Hystory of Asthma

Family Hystory of Asthma No Family Hystory of Asthma

Fig. 3. FENO values correlation with family history of asthma

DISCUSSION
Allergic rhinitis and asthma are considered a single respiratory disease, a single respiratory syndrome involving two parts.
Bronchial hyperreactivity is frequently present in patients withmoderate-severe allergic rhinitis and should be suspected in thepresence of risk factors (17).
In the present study we looked for the presence of lower
airways inflammation, a potential subclinical inflammation inpatients with allergic rhinitis using FENO, a marker of eosinophilic
inflammation. FENO is an accepted marker which increased inlower airway inflammation (18).
The present study investigate the presence of lower airways
inflammation in patients with allergic rhinitis
We obtained a correlation of FENO with sensitization, withseverity of rhinitis and family history of asthma. Our data notsuggested any association with monosensitization and polisensitization in our patient. Although in other studies it was shown, inchildren an increased of lower airway inflammation when house
dust mites sensitization is present (19,20).
Asthma risk factors should be evaluated in every patient withallergic rhinitis. Parental history of atopy or asthma is positively
associated with an increased risk for asthma (21,22). In ourstudy we found o statistical significant correlation of FENO andthe presence of asthma in family history. Patients with allergic
rhinitis and family history of asthma should be closely and more
often examine for asthma symptoms.
Although moderate-severe allergic rhinitis was seen often
associated with asthma than mild allergic rhinitis (17), in ourstudy the severity of allergic rhinitis was not correlated withFENO values. More studies should be done in order to evaluate
the progression from rhinitis to asthma.
Therefore, from daily practice point of view is important toevaluate for asthma every patient with allergic rhinitis (1).Patientswith allergic rhinitis presenting other risk factors for asthma
should be closely evaluated for subclinical lower airways inflammation which is the substrate for bronchial hyperresponsiveness.

1. Bousquet J, Khaltaev N, Cruz AA, et al: Allergic rhinitis and its impact
on asthma. Allergy, 2008; 63:7-160.
2. GINA Guide 2009. http://www.ginasthma.com/
3. The international Study of Asthma and Allergies in Childhood (ISAAC)
Steering Commitee Worldwide variation in prevalence of symptoms ofasthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. Lancet,
1998; 351: 331-334.
4. Settipane RJ, Hagy GW, Settipane GA. Long-term risk factors fordeveloping asthma and allergic rhinitis: a 23-year follow-up study ofcollege students. Allergy Proc., 1994; 15(1): 21-5.
5. Christodoupoulos P, Cameron L, Durham S, et al: Molecular pathology of allergic disease II: upper airway disease. J Allergy Clin Immunol.,
2000; 105: 211-223.
6. Bousquet J, Van Cauwenberge P, Khaltaev N: Allergic rhinitis and its
impact on asthma. J Allergy Clin Immunol, 2001; 108: S147-334.
7. Kowalski ML, Alam R. Signal transduction mechanisms as new targets
for allergists. Allergy, 2001; 56: 199-203.
8. Kharitonov S, Chung KF, Evans DJ, er al. The elevated level of exhaled nitric oxide in asthmatic patients is mainly derived from the lower
respiratory tract. Am J Respir Crit Care Med, 1996; 153: 1773-1780.
9. Horvath I, Barnes PJ. Exhaled monoxides in asymptomatic atopic
subjects. Clin Exp Allergy, 1999; 29: 1276-1278.
10. Vass G, Huszar E, Augustinovicz M, et al. The effect of allergic
rhinitis on adenosine concentration in exhaled breath condensate. Clinand Exp Allegy, 2006; 36: 742-747.
11. Prieto L, Gutierrez V, Uixera S. Exhaled Nitric Oxide and Bronchial
Responsiveness To Adenosine 5’-momophosphat in Subjects With
Allergic Rhinitis. Chest, 2002; 121: 1853-59.
12. Henriksen AH, Sue-Chu M, Lingaas H, et al. Exhaled andnasal NO levels in allergic rhinitis:relation to sensitization, pollen
season and bronchial hyperresponsiveness. Eur Resp J, 1999; 13:301-306.
13. Price D, Berg J, Lindgren A. An economic evaluation of NIOXMINO airway inflammation monitor in the United Kingdom. Allergy,
2009; 64: 431-8.
14. Barnes KC. Evidence for common genetic elements in allergic
disease. J Allergy Clin Immunol., 2000; 106(5 Suppl): S192-200.
5. Aronsson D, Tufvesson E, Bjermer L. Alleric rhinitis with or without
concomitant asthma: difference in perception of dyspnoea and levels offractional exhaled nitric oxide. Clin Exp Allergy, 2005; 35:1457-61.
16. Dreborg S. Skin test used in type I allergy testing. Position Paper.
Allergy, 1989; 44: 22-31.
17. Cirillo I, Pistorio A, Tosca M, Ciprandi G. Impact of allergic rhinitis
on asthma: effects on bronchial hyperreactivity. Allergy, 2009; 64(3):439-444.
18. Silkoff PE, Carlson M, Bourke T, et al. The Aerocrine exhaled nitricoxide monitoring system NIOX is cleared by the US Food and Drug
Administration for monitoring therapy in asthma. J Allergy Clin Immunol,
2004; 114(5): 1241-56.
19. Marcucci F, Passalacqua G, Canonica GW, et al. Lower airway
inflammation before and after house dust mite nasal challenge: anage and allergen exposure-related phenomenon. Respir Med., 2007;

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Patients’ quality of life is better influence when allergic inflammation is controlled using ARIA guideline reccomandations (23).
In conclusion FENO is an important tool to evaluate
subclinical allergic inflamationin lower airways in patient withallergic rhinitis and is correlated with other risk factors for asthma
develops in allergic patients.

34

101(7): 1600-8.
20. Inal A, Kendirli SG, Yilmaz M, et al. Indices of lower airway inflammation in children monosesitized to house dust mite after nasal allergenchallenge. Allergy, 2008; 63: 1345-51.
21. Holgate S. Genetic and environmental interaction in allergy andasthma. J Allergy Clin Immunol., 1999; 104: 1139-46.

22. Tang EA, Wiesch DG, Samet JM. Epidemiology of asthma and allergic diseases. Middleton’s Allergy Principles and Practice, ed. 6, vol.
1, ed. Mosby, 2003:1127-1168
23. Braido F, Baiardini I, Balestracci S, et al. Does asthma control
correlate with quality of life related to upper and lower airways? A reallife study. Allergy, 2009; 64: 937-43.

OXIDUL NITRIC IN AERUL EXPIRAT SI RISCUL DEZVOLTARII ASTMULUILA PACIENTII CU RINITA ALERGICA
REZUMAT
Introducere: Relaţia dintre căile respiratorii superioare şi inferioare este la ora actuală extreme de bine cunoscută. Rinita alergică
este considerată ca factor de risc pentru dezvoltarea astmului şi deasemenea este o comorbiditate în astm. Istoricul familial de astm
bronşic este cunoscut ca factor de risc în dezvoltarea astmului bronşic.
Scopul acestui studiu este de a evalua riscul de a dezvolta astm la pacienţii cu rinită alergică folosind determinarea oxidului nitricîn aerul expirat (Fe-NO).
Metodă: 34 pacienti cu rinită alergică au fost evaluaţi folosind Nioxmino pentru determinarea Fe-NO. Antecedentele heredocolaterale
au fost cu atenţieanalizate, evaluandu-se şi scorul celor cinci simptome nazale. Testele cutanate alergologice au fost efectuate fiecărui
pacient pentru alergeni inhalatori (acarieni, polen, epitelii de pisică, epitelii de câine, mucegaiuri).
Rezultate: 15 pacienti (44,11%) din lotul de studiu au fost de sex feminin (sex ratio f:m=1:1.26). Media vârstei acestor pacienţi este
28,77 ± 10,38 ani. Toţi pacienţii au fost alergici, 52,9% polisensibilizaţi la mai mult de un alergen. Am găsit o corelatie semnificativa
statistic între istoricul medical de astm broşic şi valorile crescute ale Fe-NO (p = 0,001). Dar, între pacienţii polisensibilizaţi şi monosensibilizaţi nu s-a găsit corelatie cu Fe-NO (p = 0,19), şi deasemenea nu s-a constatat o corelatie semnificativa statistic cu scorul
simptomelor rinitei (p = 0,08).
Concluzii: Valorile crescute ale Fe-No ar putea fi considerate ca factor de risc pentru dezvoltarea astmului la pacienţii cu rinită
alergică. În rinita alergică, severitatea scorului simptomelor nu poate fi considerat un factor predictiv al dezvoltarii astmului. Nu s-a
constatat nici o corelaţie a valorilor crescute ale Fe-NO şi polisensibilizare.
Cuvinte cheie: oxid nitric expirat, astm, rinita, antecedente familiale

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DRB1 ALLELE FREQUENCY IN A POPULATION GROUPFROM THE WESTERN PART OF ROMANIAELENA GAI1*, ALEXANDRA GRUIA1, FLORINA BOLDEANU1, MIRABELA CRISTEA1, DIANALUNGEANU2, MARGIT SERBAN3, SMARANDA ARGHIRESCU3, CRISTIAN JINCA3, LOREDANA BALINT GIB4, VALENTIN ORDODI5, VICTOR DUMITRASCU6, CALIN TATU5, VIRGIL
PAUNESCU7
1Regional Center for Transplant Immunology, Emergency Clinical County Hospital Timisoara
2
Department of Medical Informatics and Biostatistics, UMF “Victor Babes” Timisoara
3
Pediatrics Department, UMF “Victor Babes” Timisoara
4
Stem Cell Transplantation Department, Emergency Clinical Children Hospital “Louis Turcanu”, Timisoara
5
Department of Biology, UMF “Victor Babes” Timisoara
6
Clinical Laboratory Department, Emergency Clinical County Hospital Timisoara
7
Department of Physiology-Immunology, UMF “Victor Babes” Timisoara

ABSTRACTThe present study was conducted between 2008 and 2010 and it aimed to investigate HLA-DRB1 gene polymorphism in populationgroup from the Western part of Romania. PCR-sequence-specific oligonucleotide probes (PCR-SSO) technique was used to type
HLA-DRB1 genes. The frequencies of the alleles for male and female population were calculated and compared.
Keywords: HLA-DRB1, alleles, PCR with sequence-specific oligonucleotides (PCR-SSO) technique

INTRODUCTION
The major histocompatibility complex (MHC) encodesclass I and class II HLA molecules, and some complement
components (1,2).
The HLA complex includes almost four million base pairsof DNA, encoding the HLA-A, B, C (class I), and HLA-DR, DQ,
and DP (class II) antigens. To date, numerous studies have been
performed to study the HLA polymorphism in different populations
and ethnic groups (3).
For example, the HLA-DRB1 with its more than 315 known
alleles represents the most polymorphic protein-encoding regionsof the human genome (4,5). In addition to their role in conferring
immune identity, genetic distances and correspondence analysis
demonstrated that patterns of allele and haplotype distribution ofclass I and class II loci are racially and geographically restricted,
thereby allowing their use for population and evolution studies(6).
The aim of our work was to investigate the frequencies of the
HLA-DRB1 alleles in Romanian population. The samples wereanalyzed by the polymerase chain reaction sequence-specificoligonucleotides (PCR-SSO) method.
In this paper, the typing results for the HLA class II loci
(DRB1) in a group of 501 individuals from the Western part ofRomania are presented. The data were examined at the allele

genotype level.
MATERIALS AND METHODS
Samples
The whole blood samples were collected from 501 individuals living in the Western part of Romania between 2008 and 2010.
The studied samples were from the Emergency Children Hospital“Louis Turcanu” in Timisoara, Department of Transplant, and also
from the Blood Center of Timisoara. Samples were genotyped in
the Laboratory of Immunology of Transplant of the Emergency
County Hospital Timisoara, Romania. Informed consent was
obtained from all subjects and the experiments performed for thisinvestigation complied with current guidelines and ethics.
HLA typing
HLA typing was performed using 2 milliliter peripheral bloodcollected in ethylenediaminetetraacetic acid (EDTA) tubes forevery sample. Blood sample collection and HLA molecular typing
were carried out with the national and local ethical consent (7).
Blood was collected in order to establish the HLA genotype formedullar transplant. DNA was extracted using an automatic DNA
extractor MagNA Pure LC DNA isolation with a specific kit. The
kit contained washing buffers, lysis solutions, proteine kinase,

Received October 2010. Accepted November 2010. Address for correspondence: Elena Gai, Center for Transplant Immunology, Emergency Clinical County Hospital
Timisoara, 10 Bv. Iosif Bulbuca, Ro-300736, phone/fax: 0356433211, e-mail: elagai@yahoo.com

Fiziologia - Physiology z 2010.20.4 (68)

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suspensions with magnetic beads, elution buffers.
The DNA was resuspended in deionised water or the buffer
provided with the extraction kit, at a concentration of 13-15ng/
μL and A260/A280 ratio of 1.65-1.80.
The Dynal RELI SSO HLA-DRB1 test was based on three
processes: (i) PCR-target amplification (8,9), (ii) hybridisationof the amplified products to an array of immobilized sequencespecific oligonucleotide probes, and (iii) detection of the probebound amplified product by colour formation (10). The AutoRELI
48 Instrument automated the hybridisation and the detection
reaction. HLA–DRB1 typing was carried out with polymerase
chain reaction, using sequence-specific oligonucleotides (PCRSSO) of HLA–DRB1 allele.
In order to perform the amplification of the samples, it wasnecessary to have both a positive control, and a negative control
DNA. The following reagents were necessary: 7.5 μL (6.0 mM)
MgCl2 solution, 15 μL Mastermix, 7.5 μL DNA (concentration of
13-15 ng/μL to give total of approximately 100 ng/reaction).
The plate with samples was introduced in the PCR andunderwent a thermal cycler program: a cycle program (35 cycles)
at 95ºC for 15 sec., at 60ºC for 45 sec., at 72ºC for 15 sec., holdprogram at 72ºC for 5 min., hold program 15ºC forever.
It was necessary to pipette 30 μL of denaturizing solution into
each reaction, which was then followed by 10 min. incubation at
the room temperature, to allow a complete denaturizing.
The biotin-labeled amplicons hybridized to those SSO
probes that contained a complementary target sequence andthus were “captured” onto the membrane strip.
The amplicon probe complex was visualized using acolorimetric reaction. Streptavidin-horseradish peroxidase (SAHRP) conjugate was added to the membrane and bound to thebiotin-labelled amplicons captured by the SSO probe. Additionof hydrogen peroxide (H2O2) and tetramethylbenzidine (TMB)
substrate, results in the formation of a blue color complex in thepresence of SA-HRP.
The AutoRELI 48 Instrument automateed both the hybridisation and the detection reactions.
The data were interpreted with the software Dynal Biotech
Pattern Matching Program 5.42.
Statistical analysis
Statistical analysis of the data was performed using SPSSv. 15.0. The results for the categorical data were presented aspercentages. The Chi-square test was applied to investigate thestatistical significance of the observed differences in frequency
between the two sexes.
RESULTS AND DISCUSSIONSThe HLA-DRB1 allele frequencies in the Romanian population group (271 males and 230 females) were summarizedin Table 1. There were 501 individuals in total, with 14 types ofallele. In the locus DRB1, DRB1*11 was the most frequent allele
in the present study with a frequency of 43.91 %, followed by

DRB1*07 with a frequency of 21.76 %.
Table 1. Allele frequencies of the HLADRB1 loci in a Romanian population group ofWestern part of Romania
HLATotal
Males
Females
DRB1
N=501
N=271
N=230
allele
n
%
n
%
n
%
1
47
9.38 27 9.96 20
8.70
3
52
10.38 26 9.59 26
11.30
4
40
7.98 21 7.75 19
8.26
5
1
0.20
1
0.37 0
0.00
6
1
0.20
1
0.37 00.00
755
10.98 31 11.44 24
10.43
8
8
1.60
5
1.85 3
1.30
9
2
0.40
1
0.37 1
0.43
10
4
0.80
2
0.74 20.87
11
111 22.16 64 23.62 47
20.43
12
7
1.40
4
1.48 31.30
1351
10.18 25 9.23 26
11.30
14
25
4.99 13 4.80 12
5.22
15
49
9.78 26 9.59 23
10.00
16
46
9.18 23 8.49 23
10.00
17
1
0.20
1
0.37 0
0.00
18
1
0.20
0
0.00 1
0.43
HLA DRB1*11 was the most frequent observed allele in boththe male individuals and the female individuals, with a percentof 23.62%, and 20.43%, respectively. The difference between
male and female population was not significant (Chi-square
test, p=0.39). The allele HLA DRB1*07 had also a high percent
in male population with 11.44%, while in the female population
HLA DRB1*07 had an equivalent occurrence with DRB1*15
and DRB1*16. In female population a high percent of DRB1
allele was observed for DRB1*03 and DRB1*13with a percentof 11.30% each (none were statistically significant). Was also
observed that in the male population there were no DRB1*18
allele, while in the female population there were no DRB1*05,
DRB1*06, and DRB1*17 allele.
CONCLUSIONSThis study found that the most frequent allele in a populationgroup of 501 individuals was HLA DRB1*11 with no significant
difference between the male and female population in the Western part of Romania.
Acknowledgements. This paper was supported by the
National Program of Transplant.
REFERENCES
1. Francke U, Pellegrino MA. Assignment of the major histocompatibility complex to a region of the short arm of chromosome 6. Proc.
Natl.Acad.Sci USA, 1977; 74, 1147-1151.
2. Trowsdale J, Young JA, Kelly AP, Austin PJ, Carson S, Meunier H,

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So A, Erlich HA, Spielman RS, Bodmer J. Structure, sequence andpolymorphism in the HLA-D region. Immunol.Rev., 1985; 85, 5-43.
3. Kabbaj M, Oudghiri M, Naya A, Naamane H, El Turk J, Bennani S,
Hassar M. HLA-A, -B, -DRB1 alleles and haplotypes frequencies in
Moroccan patients with leukemia. Ann Biol Clin., 2010; 68 (3): 291-6.
4. Marsh SG, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, Geraghty DE, Hansen JA, Mach B, Mayr WR, Parham P, Petersdorf EW, Sasazuki
T, Schreuder GM, Strominger JL, Svejgaard A, Terasaki PI. Nomenclature forfactors of the HLA system. Tissue Antigens, 202; 60:407-464.
5. Robinson J, Waller MJ, Parham P, de Groot N, Bontrop R, Kennedy
LJ, Stoehr P, Marsh SG. IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic
Acids Res., 2003; 31:311-314.
6. Almawi WY, Busson M, Tamim H, Al-Harbi EM, Finan RR, WakimGhorayeb SF, Motala AA. HLA Class II Profile and Distribution of
HLA-DRB1 and HLA-DQB1 Alleles and Haplotypes among Lebaneseand Bahraini Arabs. Clinical and Diagnostic Laboratory Immunology,

2004; 11(4):770-774.
7. Feltbower RG, Manda SOM, Gilthorpe MS, Greaves MF, Parslow
RC, Kinsey SE, Bodansky HJ, McKinney PA. Detecting Small-Area
Similarities in the Epidemiology of Childhood Acute Lymphoblastic
Leukemia and Diabetes Mellitus, Type 1: A Bayesian Approach. Am J
Epidemiol., 2005; 161: 1168-1180.
8. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim
N. Enzymatic amplification of beta-globulin genomic sequences andrestriction site analysis for diagnosis of sickle cell anaemia. Science,
1985; 230:1350-54.
9. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via apolymerase-catalysed chain reaction. Methods in Enzymology, 1987;
155: 335-350.
10. Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysisof amplified DNA with immobilised sequence-specific oligonucleotide
probes. Proceedings of the National Academy of Sciences USA, 1986;
86, 6230-38.

FRECVENTA ALELEI DRB1 IN GRUPUL POPULATIONAL DIN PARTEA DE
VEST A ROMANIEIREZUMAT
Studiul prezent a fost efectuat intre anii 2008-2010 si si-a propus investigarea polimorfismului genei HLA-DRB1 in grupul populational
din partea de vest a Romaniei. In acest scop, a fost utilizata tehnica PCR-SSO pentru tipizarea genei HLA-DRB1. Au fost calculate
si comparate frecventele alelelor in cadrul populatiilor de sex masculin si feminin.
Cuvinte cheie: HLA-DRB1, alele, tehnica PCR-SSO

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LETTER TO THE EDITOR

RADIOPROTECTIVE PROPERTIES OF WATER WITH LOWCONTENT OF STABLE ISOTOPES: CRITICAL EVALUATIONSERGEI V. JARGIN
Peoples’ Friendship University of Russia, Moscow

ABSTRACT
Deuterium oxide (D2O) concentration in natural water is about 0,02 %. First symptoms appear in experimental animals when D2O
concentration in body fluids reaches 20-25 %. The stable oxygen isotope 18О is harmless even at highest enrichments. D2O and18
О concentrations are permanent environmental factors, and all living organisms are adapted to them. The concluding point is that
decreased contents of microcomponents such as D2O and 18О, which are biologically indifferent also at much higher concentrations,cannot impart useful qualities to drinking water.
Key words: deuterium, stable isotopes, radiation safety, spleen, mouse

In 1999 in the Romanian Journal of Physiology was published an article (1), where radioprotective and immunostimulating
effects of deuterium-depleted water in mice were reported. During
last years, this theme has become popular among some Russian
researchers: radio-protective properties of the „light water“ were
demonstrated in experimental animals predominantly on thebasis of histopathological analysis of the spleen. Considerable
differences in murine spleen morphology were shown between
the radiation-exposed animals receiving usual water on one side,and deuterium-depleted (2) or deuterium- and 18O-depleted water(3) on the other side. The study (3) was funded by the Federal
agency for science and innovations of Russian Federation.
Furthermore, protective effect of the deuterium- and 18O-depleted water against radiation-induced lenticular opacity in mice
was reported (4). Cataracts are a common pathological abnormality of the lens characterized by the loss of lens transparency.
They are the leading cause of blindness worldwide. Because of
its high prevalence, this disease has substantial public health
implications (5). The lens of the eye is recognized as one of themost radiosensitive tissues in the human body. Ultraviolet rays (5)and ionizing radiation (6) can induce formation of the cataracts.
Therefore, a proposition about protective effects of the ‘lightwater’ against the cataract is of great potential importance forpublic health. However, reliability of such statements is doubtful
because of the following reasons.
In the nature, the ratio of deuterium to hydrogen (D/H) is
about 1:6600, which means that concentration of deuteriumin natural water is about 150 parts per million (ppm) or 0.015
atom% (7). First symptoms appear in experimental animals at
D2O concentrations in corporal fluids of more than 20-25 %,while death follows after a concentration increase up to 30-35% or higher (8). High concentrations (usually more than 20%
of body weight) can be toxic to human and animal cells (9). In
other words, natural D2O concentration is more than 1000 times
below the threshold level. Should the “light water” have a radioprotective effect, it would mean that D2O in concentration of 0.015
atom% enhances radiation-induced damage, which has neverbeen observed even at much higher concentrations, exactly asother adverse effects were not. This issue is rather well studied
because stable isotopes are used in clinical and experimental
research (10,11), and heavy water is used in nuclear reactors.
The stable oxygen isotope 18O is harmless even at maximallypossible enrichments (10). On the contrary, there is informationon the radio-protective and other beneficial effects of water withenhanced deuterium concentration (7,9,12,13), which is not
mentioned in the articles (2-4).
D2O and 18О concentrations in water are constant environmental factors, and all biological species have been adapted
to their natural concentrations in the course of the evolution.
Besides, the technically complicated process of water clearance
from the stable isotopes (rectification with re-mineralisation) (3)can negatively influence the qualities of drinking water. Investigations of the «light water» can be compared with a study of waterdevoid of some natural microelements: prolonged consumptionof such water can cause deficiency of a microelement, but apositive effect from a lowered concentration of a normal environmental micro-component is very improbable. More probableappears adjustment of the morphological description of murine
spleen, which provided the basis for the conclusions about theradioprotective effects of the “light water”, to a preconceived
idea (14,15). For example, it is stated with regard to the spleenmorphology in radiation-exposed mice, receiving deuterium-de-Received January 2010. Accepted May 2010. Address for correspondence: Sergei V. Jargin, Clementovski per 6-82; 115184 Moscow, Russia, e-mail: sjargin@mail.ru

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pleted water (verbatim from Russian): “Morphological parametersof peri-arterial lymphoid mantles are elevated… irregularly
enhanced cell destruction in lymphoid zones of the spleen wasobserved… considerable (2-6 times) elevated contents of granular leukocytes… The quantity of cells with mitoses in germinal
centres was 6.9 times elevated” (2). Decreased concentration of
biologically indifferent micro-components, such as deuterium and18
О, could not have caused significant morphological differencesfrom the control.
Considering the above, conclusions and recommendations
on the basis of the experiments with deuterium- and 18O-depleted
water should be regarded as unfounded. The concluding point isthat decreased contents of microcomponents such as D2O and18
О, which are biologically indifferent also at much higher concentrations, cannot impart useful qualities to drinking water.
REFERENCES
1. Bild W, Stefanescu I, Haulica I, Lupuşoru C, Titescu G, Iliescu R,
Nastasa V. Research concerning the radioprotective and immunostimulating effects of deuterium-depleted water. Rom J Physiol, 1999;
36(3-4):205-18.
2. Grigorenko DE. Radio-protective influence of the light water on thelymphoid tissue of murine spleen (in Russian). Vestnik Limfol, 2008;(3):42.
3. Rakov DV, Erofeeva LM, Grigorenko DE, Abrosimova AN, Vorozhtsova
SV, Muratova MIu, Siniak IuE. The influence of the water with decreased
content of heavy stable hydrogen and oxygen (18O) isotope on development of radiation injuries at gamma-radiation in low dose (in Russian withEnglish summary). Radiats Biol Radioecol, 2006;46(4):475-9.
4. Abrosimova AN, Rakov DV, Siniak IuE. The ”light” water effect onlenticular opacity development in mice after repeated low dose gammairradiation (in Russian with English summary) Aviakosm Ekolog Med,2009;43(2):29-32.
5. Berthoud VM, Beyer EC. Oxidative stress, lens gap junctions, andcataracts. Antioxid Redox Signal, 2009, 11(2):339-53.
6. Ainsbury EA, Bouffler SD, Dörr W, Graw J, Muirhead CR, Edwards
AA, Cooper J. Radiation cataractogenesis: a review of recent studies.
Radiat Res, 2009, 172(1):1-9.
7. Somlyai G, Jancsó G, Jákli G, Vass K, Barna B, Lakics V, Gaál T.
Naturally occurring deuterium is essential for the normal growth rate ofcells. FEBS Lett, 1993, 317(1-2):1-4.
8. Puliaevskiĭ AG. Metabolic kinetics and biological action of heavy
water (D2O) (in Russian). Gig Sanit, 1986;(3):63-6.
9. Kushner DJ, Baker A, Dunstall TG. Pharmacological uses and perspectives of heavy water and deuterated compounds. Can J Physiol
Pharmacol, 1999; 77(2):79-88.
10. Jones PJ, Leatherdale ST. Stable isotopes in clinical research: safety
reaffirmed. Clin Sci (Lond), 1991, 80(4):277-80.
11. Koletzko B, Sauerwald T, Demmelmair H. Safety of stable isotope
use. Eur J Pediatr, 1997, 156 Suppl 1:S12-7.
12. Laeng RH, Mini RL, Laissue JA, Schindler R. Radioprotection ofcultured cells by preincubation in medium containing deuterium oxide.
Int J Radiat Biol, 1991; 59(1):165-73.
13. Michel F, Altermatt HJ, Gebbers JO, Bally E, Berchtold W, Laissue
JA. Radioprotection by pretreatment with deuterated water: cytokinetic
changes in the small intestine of the mouse. Virchows Arch B, 1988,
54(4):214-20.
14. Jargin SV. Radio-protective properties of water with low contentsof stable isotopes: inadequate planning of experiments (in Russian).
Ukr Med J, 2009; 73(5):102.
15. Jargin SV. Deuterium in lymphology: inadequate planning of experiments (in Russian). Vestnik Limfol, 2009; (2):36-39.

PROPRIETATILE RADIOPROTECTOARE ALE APEI CU CONTINUT SCAZUT
DE IZOTOPI STABILI: EVALUARE CRITICAREZUMAT
Concentratia oxidului de deuteriu (D2O) in apa naturala este de 0,02 %. Primele simptome apa la modelul experimental animal atuncicand concentratia D2O in fluidele organismului atinge valoarea de 20-25 %. Izotopul stabil al oxigenului 18О este inofensiv chiar si laconcentratii crescute. Concentratiile D2O si 18О sunt factori de mediu permanenti si toate organismele vii se adapteaza la acestea.
Concluzia acestui studiu este ca scaderea continutului microcomponentelor de tipul D2O si 18О, care sunt biologic indiferente, chiar
si la concentratii mult mai mari, nu poate afecta calitatile benefice ale apei potabile.
Cuvinte cheie: deuteriu, izotopi stabili, siguranta radiatiei, splina, soarece

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