FIZIOLOGIA
physiology
FOUNDING EDITORFRANCISC SCHNEIDERCHIEF EDITORCARMEN PANAITESCUCO-CHIEF EDITORSCARMEN TATUFLORINA BOJINASSOCIATE EDITORSMIHAI NECHIFORSORIN RIGAEXECUTIVE EDITORSGABRIELA TANASIEDACIANA NISTORMARIUS GEORGESCUE DI TO RI AL BOAR D
ARDELEAN AURELBĂDĂRĂU ANCA
BENEDEK GYORGYBENGA GHEORGHECOJOCARU MANOLEGĂLUȘCAN ATENAIANCAU MARIAMIHALAŞ GEORGETA
MUNTEAN DANINAMUREŞAN ADRIANANESTIANU VALERIUOPREA TUDORPANAITESCU CARMEN(Arad)(Bucureşti)(Szeged)(Cluj)(Bucureşti)(Timișoara)(Craiova)(Timişoara)(Timişoara)(Cluj)(Craiova)(New Mexico)(Timişoara)PĂUNESCU VIRGILPETROIU ANAPODARIU ANGELA CODRUTA
RÂCZ OLIVERRIGA DANSABĂU MARIUS
SAULEA I. AURELSIMIONESCU MAIASWYNGHEDAUW BERNARDTANGUAY M. ROBERTTATU ROMULUS FABIANVLAD AURELIANVOICU VICTORZĂGREAN LEON

ACCREDITED BY CNCSIS - B+CATEGORY ■ CODE 240
http://www.ebscohost.com/titleLists/a9h-journals.pdf
Fiziologia (Physiology) is issued quarterly
Printed at Editura EUROSTAMPA
www.eurostampa.ro
Bd. Revoluţiei din 1989 nr. 26, Timişoara
Tel/fax: 0256-204816

ISSN 1223-2076(Timişoara)(Timişoara)(Timișoara)(Kosice)(Bucureşti)(Tg. Mureş)(Chişinău)(Bucureşti)(Paris)(Canada)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)Instructions to Authors
Submission: Only original papers in English areconsidered and should be sent to the following
address: carmen.tatu@umft.ro
Manuscripts should be submitted by e-mail only,
written in Microsoft Word 97 or later versions.
Conditions: AII manuscripts are subject to editorial
review. Manuscripts are received with the explicit
understanding that they are not under simultaneousconsideration by any other publication. Submission ofan article for publication implies the transfer of the
Copyright from the author the publisher upon
acceptance. Accepted papers become the permanent
property of "Fiziologia" (Physiology) and may not be
reproduced by any means, in-whole or in part, without
the written consent of the publisher. It is the author's
responsibility to obtain permission to reproduce
illustrations, tables, etc. from other publications.
Arrangement:
Title page: The first of each paper should indicate
the title, the authors' names and their affiliation(s).
A short title for use as running head is also required.
Keywords: for indexing purposes, a list of 3-5
keywords in English and Romanian is essential.
Corresponding author: Indicate the full name, theemail address and the phone number.
Abstract: Each paper needs abstract and title in
Romanian and English language, fonts size 9, Arial
Narrow.
Body text: fonts size 10, Arial Narrow.
Small type: Paragraphs which can or must be set
in smaller type (case histories, test methods, etc.)
should be indicated with a „p" (petit) in the margin on
the left-hand side.
Footnotes: Avoid footnotes. When essential, they
are numbered consecutively and typed at the foot of
the appropriate page, fonts size 8, Arial Narrow.
Tables and illustrations: Tables (numbered in
Roman numerals) and illustrations (numbered in
Arabic numerals) should be prepared on separate
sheets, fonts size 9, Arial Narrow. Tables require aheading, and figures a legend, also prepared on a
separate sheet. For the reproduction of illustrations,only good drawings and original photographs can be
accepted; negatives or photocopies cannot be used.
When possible, group several illustrations on one
block for reproduction (max. size 140x188 mm) or
provide crop marks. On the back of each illustration
indicate its number, the author's name, and article
title.
References: In the text identify references by
Arabic figures, (in brackets), fonts size 9, Arial Narrow.
2

Material submitted for publication but not yet
accepted should be noted as "unpublished data" andnot be included in the reference list. The list ofreferences should include only those publications
which are cited in the text. The references should benumbered and arranged alphabetically by the authors'names. The surnames of the authors followed byinitials should be given. There should be nopunctuation signs other than a comma to separate theauthors. When there are more than 3 authors, thenames of the 3 only are used, followed by "et al"abbreviate journal names according to the Index
Medicus system. (also see International Committee ofMedical Journal Editors: Uniform Requirements formanuscripts submitted to biomedical journals. Ann
Intern Med 1982; 96: 766-771).
Examples:(a) Papers published in periodicals: Kauffman
HF, van der Heide S, Beaumont F, et al: Class-specificantibody determination against Aspergillus fumigatus
by mean of the enzyme-linked immunosorbent assay.
III. Comparative study: IgG, IgA, IgM, ELISA titers,
precipitating antibodies and IGE biding after
fractionation of the antigen. Int Arch Allergy Appl
Immunol 1986; 80:300 - 306.
(b) Monographs; Matthews DE, Farewell VT:
Using and Understanding Medical Statistics. Basel,
Karger, 1985.
(c) Edited books: Hardy WD Jr, Essex M:. FeLVinducted feline acquired immune deficiency syndrome:
A model for human AIDS; in Klein E(ed): Acquired
Immunodeficiency Syndrome. Prag Allergy, Busel,
Karger, 1986, vol 37,353 - 376.
Galley proofs: unless indicated otherwise, galley
proofs are sent to the first-named author and should
be returned with the least possible delay. Alternations
made in galley proofs, other than the corrections ofprinter's errors, are charged to the author. No page
proofs are supplied.

Fiziologia - Physiology • 2020.30.2(100)CONTENTS
Obstetrical hemorrhagic syndrome in placental insertion abnormalities …………………………………. 5
Caravețeanu DC, Moroșanu DV, Brăila AD, Brăila M
On the mechanisms underlying immunomodulatory effects of some indigenous plants ……………… 10
Sbora IN, Stănciulescu EC, Andrei AM, Bălășoiu RM, Berbecaru-Iovan A, Pisoschi CG
Significance of impaired respiratory anatomical and physiological defense mechanisms in thepathogenesis of lung disease due to nontuberculous mycobacteria (ld-ntm) in immunocompetenthosts …………………………………………………………………………………………………………………… 17
Biciusca V, Taisescu O, Stan S, Durand P, Biciusca T, Balteanu M, Mahler B
Assessment of serum levels of the anti-inflammatory cytokine il13 in obese and type 2 diabetes
patients ………………………………………………………………………………………………………………... 24Binișor ID, Moldovan RA, Moldovan I, Anghel RD, Pisoschi CG, Baniță IM
An overview on the use of probe-based confocal laser endomicroscopy in modern medical
practice………………………………………………………………………………………………………………… 34
Gheorghe AG, Anghelina F, Matei M, Demetrian AD, Olteanu M, Streba CT
Clinical Applications of Basophil Activation Test in Allergy Diagnosis …………………………………… 41
Buzan MR, Crisnic D, Grijincu M, Zbarcea LE, Tamas TP, Cotarca MD, Haidar L, Tanasie G, Panaitescu C,
Chen KW
Assessment of Cell Culture Media Variation Inducing Cytokine Secretion in PBMCs, CD3+, and CD8+
Immune Cells …………………………………………………………………………………………………………. 50
Olteanu GE, Crisnic D, Grijincu M, Cristea M, Zambori C, Ivan A, Bojin F, Gavriliuc O, Nedea E, Paunescu V
The Use of Specific Assays to Quantify Oxidative Stress in Multidrug-Resistant Bacteria…………….. 59
Tatu C, Gaspar C, Georgescu M, Simina AG, Tanasie G, Bojin F, Puscasiu D, Panaitescu C
Immunocap ISAC Multiplex Microarray IgE Testing – A Tool for Component-Resolved Diagnosis inAllergy………………………………………………………………………………………………………………….. 68
Zbircea LE, Grijincu M, Buzan MR, Gastager F, Chen KW, Panaitescu C
Post Tonsillectomy Pain in Adults – A Cohort Study …………………………………………………………. 77
Trandafir CM, Marin KC, Lupescu S, Juchiac AV, Marin HA
Interactive Pen Digitizer to Foster Teaching …………………………………………………………………… 81
Sorte SR, Rathod SB, Mohite K, Kasat P, Muthiyan G, Ahirwar A, Patil A
Evaluation of the possibility of postpartum hemorrhage in patients with placental structureabnormalities ………………………………………………………………………………………………………… 86
Caravețeanu DC, Moroșanu DV, Brăila AD, Brăila M
Development of a Miniaturized Method for Estimating Aristolohic Acid I (AAI) in Environmental
Samples ........................................................................................................................................................ 92
Draghia LP, Lukinich-Gruia AT, Zambori C, Tatu C, Tanasie G, Tatu CA, Paunescu V, Campins-Falco P,
Moliner-Martinez Y
Fiziologia - Physiology • 2020.30.2(100)3CUPRINS
Sindromul hemoragic obstetrical în anomaliile de inserție placentară ……………………………………... 5
Caravețeanu DC, Moroșanu DV, Brăila AD, Brăila M
Mecanismele care stau la baza efectelor imunomodulatoare ale unor plante indigene ......................... 10
Sbora IN, Stănciulescu EC, Andrei AM, Bălășoiu RM, Berbecaru-Iovan A, Pisoschi CG
Semnificatia alterarii mecanismelor de aparare anatomice si fiziologice respiratorii in patogeneza bolii
pulmonare datorate micobacteriilor nontuberculoase la gazdele imunocompetente ………………….. 17
Biciusca V, Taisescu O, Stan S, Durand P, Biciusca T, Balteanu M, Mahler B
Evaluarea nivelului seric al citokinei anti-inflamatoare al 13 la pacienți obezi și cu diabet zaharat de tip
2 .................................................................................................................................................................. 24Binișor ID, Moldovan RA, Moldovan I, Anghel RD, Pisoschi CG, Baniță IM
O prezentare generală a utilizării endomicroscopiei confocale laser bazate pe sondă în practica
medicală modernă ........................................................................................................................................ 34
Gheorghe AG, Anghelina F, Matei M, Demetrian AD, Olteanu M, Streba CT
Aplicațiile clinice ale testului de activare a bazofilelor în diagnosticul bolilor alergice ……………….. 41
Buzan MR, Crisnic D, Grijincu M, Zbarcea LE, Tamas TP, Cotarca MD, Haidar L, Tanasie G, Panaitescu C,
Chen KW
Evaluarea variației mediului de cultură asupra secreției de citokine a PBMCs, limfocite T CD4+ și
CD8+……………………………………………………………………………………………………………………. 50
Olteanu GE, Crisnic D, Grijincu M, Cristea M, Zambori C, Ivan A, Bojin F, Gavriliuc O, Nedea E, Paunescu V
Utilizarea de assay-uri specifice pentru cuantificarea stresului oxidativ la bacteriile
multirezistente………………………………………………………………………………………………………... 59
Tatu C, Gaspar C, Georgescu M, Simina AG, Tanasie G, Bojin F, Puscasiu D, Panaitescu C
Testarea de anticorpi IgE cu multiplex immunocap ISAC microarray – o metodă de diagnostic
molecular în alergii ………………………………………………………………………………………………….. 68
Zbircea LE, Grijincu M, Buzan MR, Gastager F, Chen KW, Panaitescu C
Durerea post-amigdalectomie la adulți – studiu cohortă …………………………………………………….. 77
Trandafir CM, Marin KC, Lupescu S, Juchiac AV, Marin HA
Stilou interactive digital pentru uz didactic …………………………………………………………………….. 81
Sorte SR, Rathod SB, Mohite K, Kasat P, Muthiyan G, Ahirwar A, Patil A
Evaluarea posibilității de hemoragie post-partum la pacienții cu anomalii de structură
placentară…………………………………………………………………………………………………………… 86
Caravețeanu DC, Moroșanu DV, Brăila AD, Brăila M
Dezvoltarea unei metode miniaturizate pentru estimarea Acidului Aristolohic I (AAI) în probe demediu ......................................................................................................................................................... 92
Draghia LP, Lukinich-Gruia AT, Zambori C, Tatu C, Tanasie G, Tatu CA, Paunescu V, Campins-Falco P,
Moliner-Martinez Y

4

Fiziologia - Physiology • 2020.30.2(100)OBSTETRICAL HEMORRHAGIC SYNDROME IN
PLACENTAL INSERTION ABNORMALITIESDRAGOȘ CRISTIAN CARAVEȚEANU1, DAN-VASILE MOROȘANU1, ANCADANIELA BRĂILA1, MIHAI BRĂILA1
1University of Medicine and Pharmacy of Craiova, Romania

ABSTRACTAim and objectives: The main purpose of this paper is to make an accurate and succinct evaluation of the obstetrical
hemorrhagic syndrome in placental insertion abnormalities. Obstetric hemorrhage is associated with an increased risk ofsevere maternal morbidity and mortality. Postpartum hemorrhage is the most common form of obstetric hemorrhage, andworldwide, a woman dies from massive postpartum hemorrhage about every few minutes.
Material and Methods: The present research study, performed both retrospectively and prospectively, was conducted
between October 2014 and March 2019, on a study group consisting of 82 selected pregnant patients diagnosed withplacental insertion abnormalities.
Results: From the point of view of the studied group, made up of the 82 patients who presented postpartum hemorrhages,
a number of 21 patients, representing a percentage of 26% had severe hemorrhages, over 1000 ml, while a number of 61
patients, representing a percentage of the studied group of 74% had minor bleeding.
Conclusions: Specific statistical analysis indicated that age, number of births, number of abortions, history of vaginal birth,weeks of pregnancy, depth of relationship between placenta and uterine muscle wall, and blood transfusion during birthwere independent risk factors for severe postpartum hemorrhage.
Keywords: obstetrical hemorrhagic syndrome, placenta praevia

INTRODUCTION
Obstetric hemorrhage is associated with an increased risk ofsevere maternal morbidity and mortality [1,2]. Postpartum
hemorrhage (PPH) is the most common form of obstetrichemorrhage, and worldwide, a woman dies from massive
postpartum hemorrhage about every 4 minutes [3,4].
In addition, after hemorrhage, many women experience
severe morbidity, such as multiple organ failure,complications of multiple blood transfusions,peripartum hysterectomy, and unintentional pelvic organ
damage, loss of fertility, and psychological sequelae,
including posttraumatic stress disorder [5,6].
The placenta is considered an important organ that evolves
with the implantation of the blastocyst throughout pregnancy[7]. This special organ plays an essential role in functions
such as nutrition, excretion, immune and endocrine function[8]. The physiological placenta is a round or oval organ,
which attaches to the uterine wall and is about 22 cm indiameter, about 2-2.5 cm thick and weighs about 1/6 of the
fetal weight at birth [9]. Thus, a normal development of theplacenta is important for a normal embryonic and fetal
development[10]. Consequently, placental abnormalities
can range from structural abnormalities to functioning
disorders to implantation abnormalities [11].
Approximately 830 women die every day worldwide as aresult of pregnancy or birth-related complications, generally
caused by obstetric hemorrhages, which remain a major
cause of maternal morbidity and mortality [12]. A systematic
review by the World Health Organization (WHO) of the
global causes of maternal deaths from 2003 to 2019 foundReceived 15th of August 2020. Accepted 19th of September 2020. Address for correspondence: Dragoș Cristian Caravețeanu, , Universityof Medicine and Pharmacy of Craiova, Romania, Petru Rares Street No. 2, Craiova; phone: 0722160462; email:cristicaraveteanu@yahoo.com

Fiziologia - Physiology • 2020.30.2(100)5that bleeding is the leading direct cause of maternal mortality
followed by hypertensive disorders [13]. Overall, bleedingaccounted for 27.1% of all maternal deaths worldwide [14].
In this context, I consider useful a study to analyze the risk
factors that were correlated with severe postpartum
hemorrhage in probably the most common placental
pathology associated with obstetric hemorrhagic
syndromes.

AIM AND OBJECTIVES
The main personal motivation for which, together with myPhD coordinator, I decided to conduct this study was that
postpartum hemorrhage is the leading cause of death inpregnant women in our country and, unfortunately, I
believe that for some cases, with a management and awell-developed and developed guide, at least some ofwhich could be saved [15].
Another very important aspect that led me to conduct thisresearch was that the placenta praevia is globally but also
nationally, in Romania, the main reason for postpartum
hemorrhage, although it has been proven that thissituation does not always lead to severe postpartum
hemorrhage [16].
Most likely, accurate assessment of the risks of severe
postpartum hemorrhage in the placenta praevia is
extremely important to reduce mortality in pregnant
women.

MATERIAL AND METHODS
The present research study, performed both
retrospectively and prospectively, was conducted between
October 2014 and March 2019, on a study groupconsisting of 103 selected pregnant patients diagnosed
with placenta praevia. Patients with surgical disease, fetal
hypoplasia, and incomplete clinical and ultrasoundinformation were excluded, resulting in 82 patients
included in the study.
The cases included in the study groups were selected fromthe case studies of the Obstetrics-Gynecology Departmentof the "Caritas" Hospital Roșiori de Vede, the private
practice of the Obstetrics-Gynecology office "Dr.
Caravețeanu Dragoș Cristian" and private practice of 2
other Obstetrics-Gynecology offices in Teleorman County.
This study was conducted entirely in accordance withexisting regulations in the field of scientific research,
provided in both European and Romanian legislation but
also according to the rules of the University of Medicine
and Pharmacy of Craiova and by approving the Ethics
Commission of the Senate of the University of Pharmacy
from Craiova.
Based on the postpartum hemorrhage management andprevention protocol issued by the Obstetrics andGynecology Associations of Europe and Romania, blood
6

loss was assessed using a combination of gauze weighing
methods and blood clot collection.
In this study, postpartum blood loss was measured at the
beginning of the skin incision for cesarean section or at the
beginning of perioperative vaginal bleeding for cesarean
section or at the beginning of parturition for vaginal birth.
Measurements were obtained for 24 hours after surgery orvaginal birth. Blood loss over 1,000 ml was defined assevere postpartum hemorrhage and was the only clinicalobservation in this study.
The diagnosis for placenta praevia was made after 28
weeks of pregnancy using ultrasonography.
Clinical suspicion of placenta praevia was maintained in
the following cases: pregnant patient with vaginal bleedingat a minimum time after 24 weeks of amenorrhea,
unprovoked associated with the absence of painful uterine
contractions, with a normal uterine tone present, with oo
dystocic presentation, with fetal heartbeats present andnormal.
Statistical processing of results and comparative data
analysis was performed using the following softwares:
Microsoft Excel version 2007, Anova One Way,
respectively SPSS version 22.0. Sublots analyze was
performed by statistical indicators: weight, median,standard deviation. Testing the consistency of the samples
was performed using the independent Student t-test,
bilateral, and testing the combination of parameters byusing the Chi square test of association. Statisticaldifferences of the test were representative when the pvalue was <0.05 at a confidence interval of 95%.

RESULTSOut of a total of 82 patients enrolled in the study, a numberof 6 had a vaginal birth, representing a percentage of
7.31%, while a number of 76 pregnant women included in
the study group, representing a percentage of 92.69 %gave birth by cesarean section, the differences being
given both by the recommendation of the attending
physician and by the guides of the Obstetrics andGynecology associations. Analyzing the data thatcharacterize the main characteristics of the study group, it
is observed that, regarding the areas of origin, apredominance of patients from urban areas, namely 61
from the study group, representing 74.39%, the mean age
was 31 years (± 12 years), the level of education wasabove the high school (post-high school studies,
bachelor's, master's) for at least 31% of the patients
included in the studied group, the duration of gestation
was in most patients included in the study under 39 weeks(over 90%) and only a number of 8 patients had over 39
weeks of pregnancy.
As associated conditions, a number of 6 patients in the
study group (representing 6.37% of the total) had highblood pressure during pregnancy, the rest of the patients
included in the study were normotensive, preeclampsia
Fiziologia - Physiology • 2020.30.2(100)was classified when blood pressure was recorded <140/90 mmHg at the first prenatal consultation, but withhypertension and proteinuria (> 0.3 g protein / 24h) afterat least 20 gestational weeks, in this case registering a
number of 14 patients from the studied group(representing 17.07% of the total).
Also, 1 patient from the studied group presented highvalues of fasting blood glucose and subsequently, by
consultation in the outpatient clinic, was diagnosed withgestational diabetes, being followed throughout
pregnancy and postpartum. From the BMI calculation,
there were values characteristic of obesity (over 30 BMI
values) in a number of 13 patients in the studied group(representing 15.85% of the total), the rest of the patients
having values of body mass index below 29.9.
Regarding the presence of abortion in the antecedents, 13
patients from the studied group (representing 15.85% of
the total) mentioned this information at the specialized
control, the rest of the patients not having an abortion in
the antecedents or not declaring this.
Also, in connection with the loss of a previous pregnancy,
4 patients from the studied group (representing 4.88% of
the total) reported the loss of a pregnancy, while 1 patientfrom the studied group (representing 1.22% of the total)
evoked the loss of two previous pregnancies. In contrast,
no patients reported losing more than two pregnancies.
From the personal pathological antecedents, it was also
highlighted that 2 patients from the studied group(representing 2.44% of the total) had venous thrombosis.
As mentioned above, out of a total of 82 patients enrolled
in the study, a number of 6 had a vaginal birth,
representing a percentage of 7.31%, while a number of 76
pregnant women included in the study group, representing
a percentage of 92.69% gave birth by cesarean section.
All of these patients were initially diagnosed with the lower
placenta inserted.
From the point of view of the studied group, made up of
the 82 patients who presented postpartum hemorrhages,
a number of 21 patients, representing a percentage of
26% had severe hemorrhages, over 1000 ml, while anumber of 61 patients, representing a percentage of thestudied group of 74% had minor bleeding. There are about
three times as many patients with severe postpartum
hemorrhage.
Regarding the age of pregnant patients included in the
study group, in the case of patients with severe
postpartum hemorrhage, the mean age was, according tostatistics, about 32 years, with extremes of 23 years and41 years, respectively, in while the age of pregnant
patients included in the study group who had minor
postpartum hemorrhage, the mean age was approximately
29 years, with extremes of 19 years and 38 years,
respectively. There is a difference of about 3 years in favorof patients who had severe bleeding, the difference being
maintained even in patients who had the extreme ages of
the group.
Fiziologia - Physiology • 2020.30.2(100)Regarding the duration of pregnancy, the statistical
calculation showed that depending on the duration ofpregnancy (more or less than 38 weeks of pregnancy) anumber of 59 patients, representing 96.72% of all those
who had minor postpartum hemorrhage (61 patients) had
a gestation period of less than 38 weeks of pregnancy,
while in the case of pregnant patients included in the study
group who had severe postpartum hemorrhage, a groupof 19 patients (90.48% of the total 21 patients) had agestation period of less than 38 weeks of pregnancy.
There is a slight difference of about 6 percent in favor ofpatients with minor bleeding, but without a strong
statistical significance. We also observed a higher
percentage in favor of patients born by caesarean section
and had major postpartum hemorrhage, both to patientswith natural birth and major postpartum hemorrhage, andto both subcategories (natural birth and cesarean section)
within patients with minor bleeding studied.
From the total number of patients studied, consulting thepatient observation sheets, with the patients' history but
also performing the clinical examination and theanamnesis with heredocolateral and personal pathologicalantecedents, we noticed that only a number of two patients(2.44% of all patients included in the group). respectively
9.52% of all patients with severe bleeding) have a historyof surgical complications in the last 15 years.
In the statistical calculations we also took into account awhole series of other clinical indices in the prediction
model of postpartum hemorrhages, among which:- history of premature birth - 2 patients with minor
hemorrhage, representing a percentage of 3.28%,
respectively a patient with severe hemorrhage,
representing a percentage of 4.76%;- history of placenta praevia - no patient with minor
hemorrhage, respectively a patient with severe
hemorrhage, representing a percentage of 4.76%;- the presence of uterine fibroids - no patient with minor
hemorrhage, respectively a patient with severe
hemorrhage, representing a percentage of 4.76%.

DISCUSSION
As previously shown, placenta praevia is a major risk
factor for severe postpartum hemorrhage. With theincrease in the number of cesarean operations worldwide,
as well as the consequent increase in the incidence of
placenta praevia, a significant increase in severe
postpartum hemorrhage has been documented. The
definition of severe postpartum hemorrhage, as mentioned
in the guidelines of obstetric and gynecological societies,
differs between different organizations and continents [5].
The definition of blood loss in severe postpartum
haemorrhage varies between different country guidelines,
ie over 1,500 mL, 2,000 mL, 30-40% of whole blood
volume or over 4 transfusion blood units (800 ml) [13].

7In this study, severe postpartum hemorrhage was defined
as blood loss over 1,000 ml. The management andprevention of postpartum haemorrhage published by the
profile associations states that when postpartum blood
loss exceeds 1,000 ml, special measures should be
implemented to save patients' lives.
These include timely referrals to a superior clinic, uterine
artery embolism or total hysterectomy, as well as intensive
care. We did not define postpartum hemorrhage based on
the volume of blood transfusion, because it is associatedwith the state of anemia, characterized by the value ofhemoglobin [16].
In this study, risk factors for severe postpartum
hemorrhage included age, number of pregnancies,
number of births, number of miscarriages, history ofvaginal birth, history of cesarean section, surgicalcomplications, body mass index before pregnancy,
number of fetuses, number of weeks at termination ofpregnancy, the presence of uterine fibroids, the types ofplacenta praevia, the depth of the placenta that invades
the uterine muscle wall and blood transfusions [17,18].
The possibility of complicated pregnancies is high and therisk of severe postpartum hemorrhage is also increased.
In addition, multiple births are considered risk factors forpostpartum hemorrhage. Pregnancy duration has also
been shown to be an independent risk factor for severe
postpartum hemorrhage. Because multiple births could
lead to changes in myometric contraction, it is thought tobe a significant factor for severe postpartum hemorrhage[13].
We found correlations with cesarean section and uterine
surgical complications with severe postpartum
hemorrhage in the placenta praevia, which was consistent
with a number of previous studies. The history of cesarean
section and uterine surgical complications are consideredrisk factors for the implantation of the placenta praevia andis frequently associated with severe postpartum
hemorrhage [19].
In addition, the body mass index calculated before
pregnancy, the number of fetuses and the presence ofuterine fibroids are associated with severe postpartum
hemorrhage, but were not independent risk factors forsevere postpartum hemorrhage. Interestingly, we foundthat a history of vaginal birth was an independent
protective factor for severe postpartum hemorrhage. This
could be explained by the subsequent relaxation of thecervix after vaginal birth.

CONCLUSION
This study suggests that placenta praevia is a major risk
factor for severe postpartum hemorrhage. With theincrease in the number of cesarean operations nationwide,
the consequent increase in the incidence of placenta
praevia has led to a significant increase in severe
postpartum hemorrhage. Specific statistical analysis
8

indicated that age, number of births, number of abortions,
history of vaginal birth, weeks of pregnancy, depth ofrelationship between placenta and uterine muscle wall,and blood transfusion during birth were independent risk
factors for severe postpartum hemorrhage.

REFERENCES
1.

World Health Organization (WHO). Maternalmortality,
15
March
2016,
http://www.who.int/mediacentre/factsheets/ fs348/en/.
2. Say L, Chou D, Gemmill A, et al. Global causes ofmaternal death: a WHO systematic analysis. Lancet Glob
Health 2014; 2(6): e323–e333.
3. Saving Lives Improving Mothers’ Care: Surveillance ofmaternal deaths in the UK 2011-13 and lessons learned to
inform maternity care from the UK and Ireland Confidential
Enquiries into Maternal Deaths and Morbidity 2009-13.
MBRRACE-UK, December 2015, https://www.npeu.ox.ac.
uk/downloads/files/mbrrace-uk/reports/MBRRACEUK%20 Maternal%20Report%202015.pdf
4. Mousa HA and Alfirevic Z. Treatment for primary
postpartum haemorrhage. Cochrane Database Syst Rev
2007; 2: CD003249.
5. Chandraharan E and Arulkumaran S. Surgical aspects ofpostpartum haemorrhage. Best Pract Res Clin Obstet
Gynaecol 2008; 22(6): 1089–1102.
6. Schuurmans N, MacKinnon C, Lane C, et al. Prevention
and management of postpartum haemorrhage. J Soc
Obstet Gynaecol Can 2000; 22: 271–281.
7. Begley CM, Gyte GML, Devane D, et al. Active versus
expectant management for women in the third stage oflabour. Cochrane Database Syst Rev 2011; 11:
CD007412.
8. Fitzpatrick KE, Sellers S, Spark P, et al. Incidence and risk
factors for placenta accreta/increta/percreta in the UK: anational case-control study. PLoS ONE 2012; 7(12):e52893.
9. Wu S, Kocherginsky M and Hibbard JU. Abnormal
placentation: twenty-year analysis. Am J Obstet Gynecol
2005; 192: 1458–1461.
10. Chandraharan E and Arulkumaran S. Management
algorithm for atonic postpartum haemorrhage. J
Paediatrics Obstet Gynaecik 2005; 31: 106–112.
11. Arulkumaran S, Mavrides E and Penney GC. Prevention
and management of postpartum haemorrhage (RCOG
Green-top Guideline No. 52 May 2009). London: RCOG
Press, 2009.
12. Condous GS, Arulkumaran S, Symonds I, et al. The
tamponade tests for massive postpartum haemorrhage.
Obstet Gynecol 2003; 101(4): 767–772.
13. B-Lynch C, Coker A, Lawal AH, et al. The B-Lynch
surgical technique for the control of massive postpartum
hemorrhage: an alternative to hysterectomy? Five cases
reported. Br J ObstetGynaecol 1997; 104(3): 372–375.
14. El-Sokkary M, Wahba K and El-Shahawy Y. Uterine
salvage management for atonic postpartum hemorrhage
using ‘modified lynch suture’. BMC Pregnancy andChildbirth 2016; 16(1): 251.
15. Ratnam LA, Gibson M, Sandhu C, et al. Transcatheter
pelvic arterial embolisation for control of obstetric andFiziologia - Physiology • 2020.30.2(100)gynaecological haemorrhage. J Obstet Gynaecol 2008;
28(6): 573–579.
16. McCandlish R, Bowler U, Van Asten H, et al. A
randomised controlled trial of care of the perineum duringsecond stage of normal labour. Br J Obstet Gynaecol
1998; 105: 1262–1272.
17. Sleep J, Grant A, Garcia J, et al. West Berkshire perineal
management trial. BMJ 1984; 289: 587–690.
18. Chandraharan E, Rao S, Belli AM, et al. The Triple-P
procedure as a conservative surgical alternative toperipartum hysterectomy for placenta percreta. Int J
Gynaecol Obstet 2012; 117(2): 191–194.
19. Teixidor Viñas M, Belli AM, Arulkumaran S, et al.
Prevention of postpartum hemorrhage and hysterectomy
in patients with morbidly adherent placenta: a cohort study
comparing outcomes before and after introduction of the
Triple-P procedure. Ultrasound Obstet Gynecol 2015;
46(3): 350–355.

SINDROMULHEMORAGICOBSTETRICALANOMALIILE DE INSERŢIE PLACENTARĂÎNREZUMAT
Scop și obiective: Scopul principal al acestei lucrări este de a realiza o evaluare precisă și succintă a sindromului
hemoragic obstetrical asociat anomaliilor de inserție placentară. Hemoragia obstetricală este asociată cu un risc crescut
de morbiditate și mortalitate maternă severă. Hemoragia postpartum este cea mai frecventă formă de hemoragie
obstetricală și, la nivel mondial, o femeie moare din cauza unei hemoragii masive postpartum aproximativ la fiecare câteva
minute.
Material și metode: Prezentul studiu de cercetare, realizat atât retrospectiv cât și prospectiv, a fost realizat între octombrie2014 și martie 2019, pe un grup de studiu format din 82 de paciente însărcinate diagnosticate cu anomalii ale inserției
placentare.
Rezultate: Din punctul de vedere al grupului studiat, format din cele 82 de paciente care au prezentat hemoragii
postpartum, un număr de 21 de paciente, reprezentând un procent de 26% au avut hemoragii severe, peste 1000 ml, întimp ce un număr de 61 de paciente, reprezentând un procent din grupul studiat de 74% au avut sângerări minore.
Concluzii: Analiza statistică specifică a indicat faptul că vârsta, numărul nașterilor, numărul avorturilor, istoricul nașterii
vaginale, săptămânile de sarcină, adâncimea invaziei placentare spre peretele muscular uterin și transfuzia de sânge în
timpul nașterii au fost factori de risc independenți pentru hemoragia severă postpartum.
Cuvinte cheie: sindrom hemoragic obstetrical, placenta praevia

Fiziologia - Physiology • 2020.30.2(100)9ON THE MECHANISMS UNDERLYING IMMUNOMODULATORYEFFECTS OF SOME INDIGENOUS PLANTSIOANA NATALIA SBORA1, ELENA CAMELIA STĂNCIULESCU1, ANA MARINA
ANDREI1, ROXANA MARIA BĂLĂȘOIU1, ANCA BERBECARU-IOVAN2,CĂTĂLINA GABRIELA PISOSCHI1
1Department of Pharmaceutical Biochemistry, University of Medicine and Pharmacy of Craiova
2Department of Pharmacology, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova

ABSTRACT
Immunity is the organism’s defense system that confers protection against diseases carried by pathogens such as bacteria,
viruses and fungi, working as a barrier against these agents. Vegetal extractive solutions (aqueous or organic) may
influence the immune system through its activity stimulation (in this case, the extractive solution has immunostimulant
properties) or depression (the extractive solution is an immunosuppressive agent). The spontaneous flora includes many
species well known for their effect on immune system, this article reviewing some cellular and physiological mechanisms
presumed to be responsible for the immunomodulatory, anti-inflammatory or anticancer potential of indigenous plants.
Keywords: immunomodulatory, innate and adaptative immune system, anti-inflammatory, indigenous plants.

INTRODUCTION
The immune system is the main defense
mechanism against infectious diseases and inflammation
produced by bacteria, fungi, viruses and other non-selfstructures. The innate immune system is the first defenseline against external pathogen agents, encountered fromthe most primitive life forms such as unicellular organisms
to mammalians, including humans (9). For humans, thefirst defense line against non-self pathogens isrepresented by biological structures like skin, mucous
membranes, enzymes (as lysozyme and phospholipase A)
present in tears and saliva, mucous present in thegastrointestinal or pulmonary tract and gastrointestinal
flora. Once pathogens have crossed these barriers,
humoral (complement system, lactoferrin, transferrin) andcellular (mast cells, natural killer cells, phagocytes, likemacrophages, neutrophils and dendritic cells) means ofdefense occur (50).
The adaptive immune system is composed of highly
specialized cells (T and B lymphocytes) that eliminate theantigen or prevent pathogens’ growth. They provide theadaptive immune system with specific properties likeimmunological memory, that leads to a stronger response
to a subsequent contact with the pathogen, and antigenic
specificity, that leads to a signal only in contact with acertain antigen (23). The protection against pathogens isassured not only through specialized cells, but also
through humoral components such as immunoglobulins
secreted by B cells (50). The immune system anomalies
can evolve to immunopathological conditions:
autoimmune diseases, inflammatory diseases and cancer(20).
Immunomodulators are agents that adjust theimmune system activity being able to prevent, ameliorate
or cure an immunological disease. They interact withdifferent parts of the immune system, so they stimulate or
inhibit different types of cells and humoral mediators
involved in the immune system, such as lymphocytes,
macrophages,
NKcells,
cytokines(36).
Immunomodulatorscanbeclassified
asimmunostimulants, drugs that increase the immunity ininfectious diseases, tumors, primary and secondary
immunodeficiency, and immunosuppressants, drugs that
decrease the immune response, so they are used in
autoimmune diseases, inflammatory diseases and organtransplant (26, 51).
Immunomodulators, especially synthetic ones,
haven’t only pharmacodynamic effects, but also
pharmacotoxicologicalones(synthetic
immunosuppressants can cause gastric irritations,
hepatitis, pancreatitis, nephropathy, myelotoxicity) (8).
Synthetic immunostimulants can also present side effects
like acute phase reaction, release of proinflammatory

Received 23rd of August, 2020. Accepted 2nd of October, 2020. Address for correspondence: Ana Marina Andrei, University of Medicine andPharmacy of Craiova, Romania; Craiova, 2 Petru Rares St., Romania; phone: 0746020457; email: marinafusaru@yahoo.com

10

Fiziologia - Physiology • 2020.30.2(100)cytokines, humoral lysis syndrome, autoimmunity and highvascular permeability with interstitial edema (44).
This is the main reason why many researches
are focused on pharmaceutical products of vegetal origin.
Vegetal immunomodulators may increase or
decrease both the innate and adaptative immune
response, but unlike those obtained through synthesis,
they are more affordable (especially the endemic ones)
and many of them with lower pharmacotoxicologicalpotential (1). Because of these advantages, themodulation of the immune response through plants
products represents a highly studied subject byresearchers from all around the world.
This paper’s aim is to review aspects regarding
plants from the spontaneous flora discussed for theirimmunomodulatory potential, mainly the active
compounds, their mechanism of action, diseases that canbe treated and cured using vegetal extracts.
We will shortly present the main indigenous families
containing plants discussed for their immunomodulatory
potential.
Aristolochiaceae family includes well known
plants, such as Aristolochia clematitis, birthwort, a ruderal
plant whose aerial part contain aristolochic acid, asubstance when used for external purposes, in small
potential, used to relief the symptomatic pain in arthritis (2).
Its mechanism of action is common with the one of thenonsteroidal anti-inflammatory drugs, at a dose of 10
mg/ml inhibiting both subtypes of cyclooxygenase (COX)(40).

doses, presents anti-inflammatory properties (in folk
medicine it is used to treat arthritis, rheumatism, gout,
chronic inflammatory and dermatological conditions), but
in high doses, due to continuous internal use it may be
carcinogenic, particularly at kidney level (29). That is why
this is a high interest subject for researchers worldwide,
they even talk about an endemic Balkan nephropathy, asa consequence of the internal administration of A.
clematitis for a long period of time, that affect lots of rural
populations (48). The anti-inflammatory mechanism ofaristolochic acid consists in phospholipase A2 inhibition(after i.m. /i.p. administration), so it inhibits the synthesisof pro-inflammatory prostaglandins (41). A double-blind
study realized on healthy volunteers has shown that
aristolochic acid increases peripheral granulocytes’
phagocytic activity after administration of a 0.9 mg/day
dose, for ten consecutive days, highlighting its
immunostimulant activity (32).
Another representative plant from this family is
Asarum europaeum L., hazelwort, plant that is commonly
found in deciduous forests. The rhizome and aerial partsare used for external purposes because they have a highconcentration of volatile oil rich in azarone, aphenylpropanoid compound with anti-inflammatory
Results of the main researches regarding themechanism of action for some indigenous plants of the13000 species of the Asteraceae family are presented inTable 1.

Table 1. Potential mechanisms of theimmunomodulatory
action for some representative plantsof the Asteraceae familySpeciesPart
usedType ofextract

Mechanism of action

Achillea
millefolium
L.
(Yarrow)

Flowers

Aqueous

In vitro, on LPS stimulated
RAW 264.7 macrophages:- Decrease of NO
synthesis and iNOS
expression;- Increase of IL-1β and
TNF-α synthesis (12)Arctium
lappa L.
(Burdock)

Roots

ButanolicIn vitro, on RBL-2H3 cells:- Decrease of IL-4 and IL-5
synthesis;- Decrease of NF-kB
synthesis (46)Fiziologia - Physiology • 2020.30.2(100)ArnicamontanaL.
(Mountain
arnica)Flowers

Hydroalcoholic

In vivo experiment on
Wistar rats:- Increases the B and T
cells synthesis;- Increase of ICAM-1
synthesis (28)Artemisia
absinthium
L.
(Absinthe)

LeavesAqueous

In vivo, on rats:-Decreases the synthesis
of IL-1 and TNF-α (6)Calendula
officinalis
L.
(Marigold)

Flowers

Aqueous

In vitro, on tumor cells
lines:-Inhibits proliferation oftumor cells (leukemia,
fibrosarcoma, breast
cancer, pancreatic, lung,colorectal cancer);
In vivo:-Growth inhibition ofmelanoma cells (25)11

Cichorium
intybus L.
(Chicory)

Flowers

EthanolicIn vivo, on rats:-Stimulates the activity of
NK cells;-Increase of IFN-γ
synthesis;-Increase of phagocytic
activity (31)Inulahelenium
L.
(Horse
heal)

Roots

EthanolicIn vitro, on alveolar
macrophage NR 8383:-Decreases the synthesis
of IL -1,-6, TNF-α (19)Matricaria
recutita L.
(Chamomile)Flowers

Aqueous

In vitro:-Inhibits COX and 5-LOX
activity, decreasing thelevels of prostaglandins
and leukotrienes;
In vivo, on rats’ skin:-Antiinflammatory effects

Colchicine is a protoalkaloid well known for its
immunomodulatory and antitumor activity, that is thereason why it is being used in rheumatic diseases, gout,
Mediterranean fever and malign conditions. It is isolated
from Colchicum autumnale’s seeds, commonly known as
autumn crocus, an indigenous Colchicaceae family’s plant.
Colchicoside and demecolcine (the later anantimitotic less toxic, isolated from bulbs and flowers, used
as a cytostatic, Colcemid, in myeloid leukemia) are
isolated from crocus the same (34). All these alkaloids
inhibit tubulin polymerization, so they block cellulardivision and also leukocytes’ migration to urate crystals
and phagocytosis (43).
Hippophae rhamnoides L., sea buckthorn,
member of the Elaeagnaceae family, is one of the mostappreciate plant with immunomodulatory potential andTable 2. Potential mechanisms of theimmunomodulatory action for some
representative plants of the Fabaceae familySpeciesPart
usedType ofextract

Mechanism of action

Baptisia tinctoria
L.
(Wild indigo)

Roots

Aqueous

In vitro, in cultures ofalveolar macrophages:- Activates macrophages
and increase IL-6 andTNF-α production (13)12against inflammation
produced by croton oil (14)Tagetespatula L.
(Frenchmarigold)

Flowers

Aqueous

In vitro:-Decreases the synthesisof oxygen reactive species;-Decreases CD4+ T cells
synthesis;-Decreases TNF-αsynthesis;
In vivo, on rats:-Decreases IL-1βsynthesis (22)Taraxacum
officinale
L.
(Dandelion)

Aerialparts

Methanolic

In vitro, on RAW 264.7
macrophages:-Decreases the synthesis
of TNF-α, IL-1β, -6, PGE2
(33)

grow spontaneous in Romania. Its fruits are rich invitamins (ascorbic acid, tocopherols, carotenoids),
flavonoids, carbohydrates, proteins, some organic acids(palmitoleic and palmitic acids identified in the mesocarp),
fruits oil being used for wound healing in the traditional
medicine, based on the anti-inflammatory, cicatrizing andrejuvenating properties (49). As it results from studies that
evaluate fruits and leaves extracts effect on
proinflammatory cytokines production (IL-2, -4, IFN-γ),
antibodies level and antioxidant potential, one can assume
that not only sea buckthorn fruits have immunomodulatory
potential but also the leaves (17).
Fabaceae family counts numerous indigenous
species discussed for their immunomodulatory properties(Table 2).

Galegaofficinalis L.
(Galega)

Aerialparts

Dichloromethane

In vitro:- Inhibits NO synthesis in
RAW
264.7
macrophages stimulatedwith LPS;- Cytotoxic activity on A549
pulmonaryadenocarcinoma cells
and U-87 glioblastoma(27)

Fiziologia - Physiology • 2020.30.2(100)Glycyrrhiza
glabra L.
(Licorice)

Roots

Aqueous

In vivo, in rats:- Inhibits colon carcinoma
cell growth;- Activates CD4+ andCD8+ lymphocytes;- Increase of antitumoral
factors synthesis (IL-2,6,-7);- Decrease of TNF-αsynthesis (7)Malvaceae family counts over 4200 plant
species, some of them well known for their antiinflammatory, immunomodulatory, expectorant andantibacterial effects. One of these is Althaea officinalis L.
studied for its immune stimulant and anti-inflammatory
activity because its great content of mucilage andflavonoids. In vitro studies have been proved that roots
aqueous extract stimulates phagocytosis and leukotrienes
release from neutrophils, as well as the release of Il-6 andTNF-α cytokines from human monocytes (5). Hypolaetin8-glucoside have proved its anti-inflammatory andanalgesic effects on rats, being even more potent thanequal doses of phenylbutazone in the suppression of
acute phase carrageenan-induced inflammation without
producing gastric ulcerations, unlike phenylbutazone.
Hypolaetin-8-glucoside have proved to be more potent
than troxerutin in decreasing the capillary permeability
induced by histamine in rats (16).
Another immunostimulatory plant is Nigella sativa L.
(black caraway), from the Ranunculaceae family, whose
seeds are widely used mainly in respiratory viral infections,
thanks to the high level of quinines (thymoquinone,
thymohydroquinone, dithymoquinone and thymol) (3).
They also contain α-hederin, an anticarcinoma saponin,
whose efficiency have been proved in vitro, on A549
(pulmonary cancer), Hep2 (laryngeal carcinoma), HT-29(colon cancer) and MIA PaCa-2 (pancreatic cancer) cells(45). It has been proved that aqueous extract of N. sativa
stimulates splenocytes proliferation in a dose dependent
manner and also the antitumoral activity of NK cells
against lymphoma YAC1 cells. It also inhibited
macrophages secretion of IL-6, TNF-α, NO, Th1 cytokines(IFN-γ, IL-2) and also it increased the secretion of Th2 antiinflammatory cytokines (IL-4, -5, -6, -10) (38). N. sativa
extracts have been intensively studied not only for theirimmunomodulatory effects, but for the antioxidant,antihistamine, anthelminthic, anticancer, antibacterial,
antihypertensive, hepatoprotective, uricosuric, antidiabetic
and anti-sterility effects (3).
Among the plants of the Papaveraceae family we
can name Chelidonium majus L., also called greater
celandine, a spontaneous Romanian plant often used for
its therapeutic properties. Both the aerial parts and the
Fiziologia - Physiology • 2020.30.2(100)Trigonella
foenum grecum
L. (Fenugreek)

Seeds

EthanolicIn vitro, on monocytes
cultures:Decreaseofproinflammatory
cytokines (IL-1α,-1β, -2, 6, TNF-α) levels;
Decrease
lipid
peroxidation,
malondialdehyde level;- Increase of SOD and
CAT activity, GSH level(10).

roots, from which there are realized crude extracts or
purified products, are being used for medical purposes.
Their therapeutic benefits (among which antiinflammatory, antioxidant, immunomodulatory andantitumoral) are related to the high level of isoquinoline
alkaloids such as chelidonine, sanguinarine, berberine(18). Their immunomodulatory potential has been provedin a plethora of in vitro studies (a protein-boundpolysaccharide extracted from C. majus showed mitogenic
activity on spleen and bone marrow cells and increased
the number of granulocyte macrophage-colony forming
cells) (47) and also in vivo studies (C. majus methanolic
extract decreased the production of TNF-α, IL-6, IFN-γ, B
cells and increased the proportion of CD4+ and CD25+
regulatory T cells in collagen induced arthritis in mice)(35). It is well known that C. majus has anticancer activity,
due to the high concentration of the compounds
mentioned above and also due to the lectins andpolysaccharides that inhibit cell growth, too (18). The
anticancer mechanism of action is complex: i) reductionof telomerase activity by chelidonine; ii) induction ofcancer cell death by apoptosis and iii) arrest of mitosis
through inhibition of tubulin polymerization (42). Ukrain™
(an anticancer drug whose major components arechelidonine, sanguinarine and chelerythrine alkaloids) is awell-known therapy successfully used in different types ofcancer, such as breast, pancreatic, ladder, or colorectal
cancer (18).
We have to mention plants of the Salicaceae family,
like Salix alba L., white willow, from whose bark was firstisolated salicine, a glycoside with anti-inflammatory,
analgesic, antipyretic properties, widely used forrheumatic diseases, the forerunner of modern
nonsteroidal anti-inflammatory medicines like aspirin (24).
Its anti-inflammatory potential has been proven in vitro (itdecreased the synthesis of PGE2, NO, TNF-α, activatedthe apoptosis of monocytes that produce proinflammatory
cytokines) (11). Also, Populus sativa buds containsaligenol, compound with COX2 inhibitory potential. The
aqueous extract of Populus gemma buds also has antiinflammatory potential, demonstrated in various animalmodels, like carrageenan rat paw edema, arachidonic acidand xylene ear edema, cotton granuloma (52).
13Mistletoe, Viscum album L., a hemiparasite
indigenous Santalaceae family plant, presents aremarkable immunomodulatory and cytostatic activity due
to its high content of lectins. It also contains viscotoxins,
small proteins with antifungal properties, enzymes, aminoacids, thiols, amines, polysaccharides, phytosterols,triterpenes, flavonoids, phenylpropane and minerals (15,39). Both the mistletoe extract and its constituents
present antitumor activity through different mechanisms: i)cytotoxic activity (lectins have cytotoxic activity on
multidrug resistant cancer cells, as colon cancer cells); ii)
increase of other anticancer drugs activit; iii) apoptosis
induction and iv) interference with tumoral angiogenesis(30). Due to its immunostimulatory properties (either invitro or in vivo, it activates the immune system’s cells andinduces anti-inflammatory cytokines) and also cytotoxic
activity, mistletoe extract is successfully used as a therapy
for patients with cancer (Isorel, Iscador, Helixor) increasing
the quality of life and presenting the advantage of rare or
mild side effects, such as nausea (39).
Another indigenous plant, Urtica dioica L.,
common nettle, a native Romanian plant from Urticaceae
family, is well-known for its therapeutic properties; its
leaves and roots have demonstrated anti-inflammatory
and cytostatic activity, due to the contained agglutinins andlectins that have mitogenic action on T cells, especially on
CD4+ helper and CD8+ cytotoxic subtypes (37). It has
also been shown that it inhibits NO synthesis inmacrophages stimulated with lipopolysaccharides (21).
Also, flavonoid glycosides isolated from aerial parts have
proved high chemotactic effect and intracellular killing
activity on neutrophils (4).

2.

3.
4.
5.
6.

7.

8.

9.
10.

11.

CONCLUSION
Indigenous plants represent a wide source ofcompounds with anti-inflammatory, immunomodulatory
and cytostatic properties. They exert these effects throughcomplex mechanisms controlling the balance between
immune cells and cells that aggress the normal bodyfunctioning. Many times, they are more used thansynthetic drugs because they don’t produce side effects,
they are more affordable and show synergic action withsynthetic drugs. So, immunomodulatory plants will always
be an intense studied topic, not only in oriental
underdeveloped countries with limited access to classic
treatment, but in western developed countries, too.

12.

13.

14.
15.

REFERENCES
1.

14

Aboughe-Angone S, Nguema-Ona E, Boudjeko T,
Driouich A: Plant cell wall polysaccharides:
Immunomodulators of the immune system and sourceof natural fibers. Curr Top Phytochem 2011;10:1-16;

16.

Adams M, Berset C, Kessler M, Hamburger M:
Medicinal herbs for the treatment of rheumatic
disorders — A survey of European herbals from the16th and 17th century. J Ethnopharmacol
2009;121:343–359;
Ahmad A, Husain A, Mujeeb M et al: A review ontherapeutic potential of Nigella sativa: A miracle herb.
Asian Pac J Trop Biomed 2013;3(5):337-352;
Akbay P, Basaran A, Undeger U, Basaran N. In vitro
immunomodulatory activity of flavonoid glycosides
from Urtica dioica L. Phytother Res 2003; 17(1):34-37.
Al-Snafi AE: The Pharmaceutical Importance of
Althaea officinalis and Althaea rosea: A Review. Int J
Pharmtech Res 2013; 5(3):1378-1385;
Amat N, Upur H, Blazekovic B: In vivo
hepatoprotective activity of the aqueous extract ofArtemisia absinthium L. against chemically andimmunologically induced liver injuries in mice. J
Ethnopharmacol 2010; 131(2):478-484;
Ayeka PA, Bian Y, Githaiga PM, Zhao Y. The
immunomodulatory
activities
oflicoricepolysaccharides (Glycyrrhiza uralensis Fisch.) in CT
26 tumor-bearing mice. BMC Complement Altern Med
2017; 17(1):536;
Bascones-Martinez A, Mattila R, Gomez-Font R,
Meurman JH: Immunomodulatory drugs: Oral andsystemic adverse effects. Med Oral Patol Oral Cir
Bucal 2014; 19(1):24-31;
Baxter D. Active and passive immunity, vaccine
types, excipients and licensing. Occup Med 2007;
57:552–556;
Bin-Hafeez B, Haque R, Parvez S et al:
Immunomodulatory effects of fenugreek (Trigonella
foenum graecum L.) extract in mice. Int
Immunopharmacol 2003; 3(2):257-265;
Bonaterra GA, Heinrich EU, Kelber O et al: Antiinﬂammatory effects of the willow bark extract STW
33-I (Proaktiv®) in LPS-activated human monocytes
and differentiated macrophages. Phytomedicine 2010;
17:1106-1113;
Burk DR, Cichacz ZA, Daskalova SM: Aqueous
extract of Achillea millefolium L. (Asteraceae)
inflorescences suppresses lipopolysaccharideinduced inflammatory responses in RAW 264.7
murine macrophages. J Med Plants Res 2010; 4(3):225-234;Classen B, Thude S, Blaschek W et al:
Immunomodulatory effects of arabinogalactanproteins from Baptisia and Echinacea. Phytomedicine
2006; 13(9-10):688-694;
Dawid Pać R: Medicinal plants used in treatment ofinflammatory skin diseases. Postepy Dermatol Alergol
2013;30(3):170–177;
Elluru S, Duong van Huyen JP, Delignat S et al:
Induction of maturation and activation of humandendritic cells: A mechanism underlying the beneficial
effect of Viscum album as complementary therapy incancer. BMC Cancer 2008; 8:161;
European Medicines Agency. Evaluation of Medicinesfor Human Use. Assessment report on Althaea
officinalis
L.
Radix.Doc.
Ref.:
MEA/HMPC/98718/2008, London, 14 May 2009;
Fiziologia - Physiology • 2020.30.2(100)17. Geethaa S, Sai Rama M, Singh V et al: Antioxidant
and immunomodulatory properties of seabuckthorn(Hippophae rhamnoides) — an in vitro study. J
Ethnopharmacol 2002; 79(3):373-378;
18. Gilca M, Gaman L, Panait E et al: Chelidonium majus- an integrative review: Traditional knowledge versus
modernfindings.
Forsch
Komplementmed
2010;17(5):241-248;
19. Grigore A, Neagu G, Dobre N et al:
Immunomodulatory potential of Inula helenium L.
Analele Stiint Univ Al I Cuza Iasi, Sect II a Biol veget
2016;62(2): 29-33;
20. Grivennikov SI, Greten FR, Karin M: Immunity,
Inflammation, and Cancer. Cell 2010; 140(6):883-899;
21. Harput US, Saracoglu I, Ogihara Y: Stimulation oflymphocyte proliferation and inhibition of nitric oxide
production by aqueous Urtica dioica extract.
Phytother Res 2005; 19(4):346-348;
22. Jabeena A, Mesaikab MA, Simjeec SU et al: AntiTNF-α and anti-arthritic effect of patuletin: A rare
flavonoid from Tagetes patula. Int Immunopharmacol
2016; 36:232-240;
23. Janeway CA Jr, Travers P, Walport M et al:
Immunobiology: The Immune System in Health andDisease. 5th edition, New York, Garland Science,
2001;
24. Jassem G: Medicinal potential of willow: A chemical
perspective of aspirin discovery. J Saudi Chem Soc
2010; 14(3):317-322;
25. Jiménez-Medina E, Garcia-Lora A, Paco L et al: A new
extract of the plant calendula officinalis produces adual in vitro effect: cytotoxic anti-tumor activity andlymphocyte activation. BMC Cancer 2006; 6:119;
26. John W: Immunostimulants. Immunol Today 1993;
14(6):275-280;
27. Karakas FP, Turker AU, Karakas A, Mshvildadze V:
Cytotoxic, anti-inflammatory and antioxidant activities
of four different extracts of Galega officinalis L (Goat’srue). Trop J Pharm Res 2016; 15(4):751-757;
28. Kawakami AP, Sato C, Neves Cardoso T, Villano
Bonamin L: Inflammatory Process Modulation by
Homeopathic Arnica montana 6CH: The Role ofIndividual Variation. Evid Based Complement Alternat
Med 2011; ID 917541;
29. Ke W, Chenchen F, Chenggang L et al: BaicalinProtects Mice from Aristolochic Acid-Induced Kidney
Injury by Induction of CYP1A through the Aromatic
Hydrocarbon Receptor. Int J Mol Sci
2015;16(7):16454–16468;
30. Kienle GS, Glockmann A, Schink M et al: Viscum
album L. extracts in breast and gynaecologicalcancers: a systematic review of clinical and preclinicalresearch. J Exp Clin Cancer Res 2009; 28(1):79;
31. Kim JH, Mun YJ, Woo WH et al: Effects of the ethanol
extract of Cichorium intybus on the immunotoxicity by
ethanol in mice Int Immunopharmacol 2002; 2(6):733744;
32. Kluthe R, Vogt A, Batsford S: Double blind study ofthe influence of aristolochic acid on granulocyte
phagocytic
activity
Arzneimittelforschung
1982;32(4):443-445;

Fiziologia - Physiology • 2020.30.2(100)33. Koh Y.J., Cha D.S., Ko J.S. et al. Anti-inflammatory
effect of Taraxacum officinale leaves onlipopolysaccharide-induced inflammatory responses
in RAW 264.7 cells J Med Food. 2010; 13(4):870-8;
34. Kuzell W, Schaffarzick RW, Naugler WE: The effectof intravenous demecolcine (colcemid) on acute gout.
AMA Arch Intern Med 1955; 96(2):153-156;
35. Lee YC, Kim SH, Roh SS et al: Suppressive effects of
Chelidonium majus methanol extract in knee joint,
regional lymph nodes, and spleen on collageninduced arthritis in mice. J Ethnopharmacol 2007;
112(1): 40–48;
36. Lee SJ, Chinen J, Kavanaugh A: Immunomodulator
therapy: monoclonal antibodies, fusion proteins,
cytokines, and immunoglobulins. J Allergy Clin
Immunol 2010;125:S314–323;
37. LeMoal MA, Colle JH, Galelli A, Truffa-Bachi P:
Mouse T-lymphocyte activation by Urtica dioica
agglutinin: II. — Original pattern of cell activation andcytokine production induced by UDA. Res Immunol
1992; 143(7):701-709;
38. Majdalawieha AF, Hmaidana R, Ronald I: Nigellasativa modulates splenocyte proliferation, Th1/Th2
cytokine profile, macrophage function and NK antitumor activity. J Ethnopharmacol 2010; 131(2): 268275;
39. Marvibaigi M, Supriyanto E, Amini N et al: Preclinicaland Clinical Effects of Mistletoe against Breast
Cancer. BioMed Res Int 2014; 2014: 785479;
40. Momin RA, de Witt DL, Nair MG: Inhibition of
cyclooxygenase (COX) enzymes by compounds from
Daucus carota L. Seeds. Phytother Res 2003, 17(8):976–979;
41. Moreno JJ: Effect of aristolochic acid on arachidonic
acid cascade and in vivo models of inflammation.
Immunopharmacol 1993; 26(1):1-9;
42. Noureini SK, Wink M: Transcriptional down regulationof hTERT and senescence induction in HepG2 cellsby chelidonine. World J Gastroenterol 2009;15(29):2603–3610;
43. Nuki G: Colchicine: its mechanism of action andefficacy in crystal-induced inflammation. Curr
Rheumatol Rep 2008;10:218–227;
44. PonceR:
Adverseconsequencesof
immunostimulation. J Immunotoxicol 2008; 5(1):3341;
45. Rooney S, Ryan MF: Effects of Alpha-hederin andThymoquinone, constituents of Nigella sativa, on
Human Cancer Cell Lines. Anticancer Res 2005;
25:2199-2204;
46. Sohn EH, Jang S, Joo H et al: Anti-allergic and antiinflammatory effects of butanol extract from Arctium
Lappa L. Clin Mol Allergy 2011; 9:4;
47. Song JY, Yang HO, Pyo SN et al: Immunomodulatory
activity of protein-bound polysaccharide extracted
from Chelidonium majus. Arch Pharm Res 2002;
25(2):158–164;
48. Stiborová M, Arlt VM, Schmeiser HH: Balkan endemic
nephropathy: an update on its aetiology. Arch Toxicol
2016; 90(11):2595-2615;

15

49. Thomas SC, Schroender WR: Sea Buckthorn(Hippophae rhamnoides L.): A multiple purpose plant.
Hort Technol 1996; 6(4):370-380;
50. Turvey SE, Broide DH: Chapter 2: Innate Immunity. J
Allergy Clin Immunol 2010; 125(2 Suppl 2): S24-S32;

51. Wiseman AC: Immunosuppressive Medications. Clin
J Am Soc Nephrol 2016; 11(2):332-343;
52. Xu Q, Wang Y, Guo S et al: Antiinflammatory and analgesic
activity of aqueous extract of Flos populi. J Ethnopharmacol
2014;152(3):540-545

MECANISMELE CARE STAU LA BAZA EFECTELORIMUNOMODULATOARE ALE UNOR PLANTE
INDIGENE
REZUMAT
Imunitatea este mecanismul de apărare a organismului ce îi conferă protecție împotriva îmbolnăvirii, acționând ca o barieră
împotriva agenților patogeni, precum bacterii, fungi, virusuri, astfel încât organismul nu contactează afecțiunea mediată derespectivul agent. Soluțiile extractive vegetale (apoase sau organice) pot influența sistemul imun, fie prin stimularea
activității sale, având efect imunostimulator, fie prin deprimarea activității acestuia, în acest caz vorbindu-se despre extracte
cu potențial imunosupresor. Flora spontană prezintă numeroase specii recunoscute pentru potențialul imunomodulator,
scopul acestui articol fiind de a prezenta unele mecanisme celulare și fiziologice presupuse a fi responsabile de potențialul
imunomodulator, antiinflamator și antitumoral al unor plante indigene.
Cuvinte cheie: imunomodulator, sistem imun înnăscut și dobândit, antiinflamator,

16

Fiziologia - Physiology • 2020.30.2(100)SIGNIFICANCEOFIMPAIREDRESPIRATORYANATOMICAL AND PHYSIOLOGICAL DEFENSEMECHANISMS IN THE PATHOGENESIS OF LUNGDISEASE DUE TO NONTUBERCULOUS MYCOBACTERIA(LD-NTM) IN IMMUNOCOMPETENT HOSTSBICIUSCA VIOREL1 ,⃰ OANA TAISESCU2*, SORINA STAN3, PATRICIADURAND4, BICIUSCA TEODORA5, MARA BALTEANU6, BEATRICE
MAHLER7
1Department of Internal Medicine, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania.
2Department of Anatomy, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania.
3Department of Internal Medicine, Resident Ph, Emergency County Clinical Hospital Craiova, Romania.
4Department of Internal Medicine, Resident Ph, ”Filantropia”Municipal Clinical Hospital Craiova, Romania.
5 Student, ”Goethe” University, Faculty of Medicine, Frankfurt am Main, Germany.
6Department of Pneumology, Faculty of Medicine, ”Titu Maiorescu” University, ”Prof. Dr. Marius Nasta” Institute for Pneumology,

Romania.
7 Department of Pneumology, Faculty of Medicine,”Carol Davila” University of Medicine and Pharmacy Bucharest, ”Prof dr Marius
Nasta” Institute for Pneumology, Romania.
⃰ These authors have equal contribution

ABSTRACT
Currently, lung disease due to nontuberculous mycobacteria (LD-NTM) can become a public health problem dueto the increasing proportion of immunocompromised people in society, on the one hand by increasing the incidenceof HIV-AIDS infection, but especially by the widespread use of antineoplastic chemotherapeutics and monoclonal
antibodies, anti-TNF and anti-IFN. As a result, the costs of diagnosing and hospitalizing these patients will be
increased, putting financial pressure on public health systems in developed countries, but especially in developing
ones. Also, the main goal of doctors is the early and correct diagnosis of these lung infections, especially sincenontuberculous mycobacteria are ubiquitous and can contaminate any sample of pathological product collectedfrom the patient.
Keywords: respiratory system defenses, NTM, immunocompetent hosts.

INTRODUCTION
If in the past, tuberculosis and leprosy were two
common human diseases caused by mycobacteria,

now bacterial infections caused by nontuberculous
mycobacteria (NTM) tend to become the main
mycobacterial diseases in developed countries, in partdue to the occurrence of HIV-AIDS infection [1] andReceived 27th of August 2020. Accepted 29th of September 2020. Address for correspondence: Balteanu Mara, PhD, ”Titu
Maiorescu” University, Faculty of Medicine, Department of Pneumology, Gheorghe Petrascu Street no 67A, RO031593 Bucharest, Romania, phone:+40213243013, email: mara.balteanu@prof.utm.roFiziologia - Physiology • 2020.30.2(100)extensive use of immunosuppressive therapeuticmeans. NTMs include saprophytic and opportunistic
microorganisms
other
than
Mycobacterium
tuberculosis and M leprae. The genus Mycobacterium
contains over 200 species and 13 subspecies, which
are grouped into 3 major groups: the Mycobacterium
tuberculosis complex, M leprae and mycobacteriaother than M. tuberculosis and M leprae(nontuberculous mycobacteria: NTM or atypicalmycobacteria)[2-4].
Atypicalmycobacteria,morphologically similar to the Koch bacillus (acid-fastbacillus: AFB), however, differ from it by faster
cultivation and sometimes at lower temperatures [5].
NTMs are ubiquitous organisms found inenvironmental sources that include drinking andnatural water, as well as soil and dust [6]. Humansubjects can inhale or ingest NTMs from water,
aerosols or dust. NTMs are opportunistic
microorganisms, in the sense that they produce
diseases only in people who have pre-existing
conditions of the respiratory system or the immune
system [7]. Several predisposing factors for lung NTM
infections are known, such as old age,
immunodepressive diseases, and drugs [8]. The
occurrence of lung disease (LD) caused by NTM (LDNTM) is the result of the interaction between: exposure
to the environment (example: infectious dose of
nontuberculous
mycobacteria,
numberofmicroorganisms and duration of exposure),
microorganisms (virulence and pathogenicity) andhost (genetic risk factors, pre-existing lung diseases
and local and systemic immune status) [9-13]. The
host organism provides antibacterial resistancethrough the anatomical defense mechanisms (thecomplex system of filtering inhaled air, respiratory
epithelial integrity, mucociliary clearance) andphysiological (inflammatory and immune reactions) of
the respiratory system [14].

RESPIRATORY SYSTEM DEFENSES
1.The general, non-specific pulmonary defense
mechanisms (first defense barrier) include
anatomical (respiratory anatomical filter, mucociliary
clearance, mucus), functional (cough and sneezing),
chemical (mucus, lysozyme, fibronectin), andbiological mechanisms (respiratory microbiome) [15].
The anatomical and mechanical filter against
microparticles, represented by the sinuous path of the
airways, consisting of the polyseptal, polycavitary
structure and dichotomous branching of the bronchi iscomplex. By passive diffusion, only the microparticles
between 0.5 and 2 μm penetrate the bronchioles andalveoli [16]. Epithelial cells together with the
mucociliary system represent a physico-chemical andbiological barrier against microbial invasion. Epithelial
18

cells are joined together by intercellular structures
called junctions, the most important structures are thetight junctions that ensure epithelial integrity [17]. The
mucociliary apparatus, made up of mucus andvibrating cilia, is the main system of physicalpurification of particles entering the airways [18]. The
mucus of the respiratory tract is viscous, has acomplex composition, the most important components
being mucins [19]. The cilia have a vibration of
1.000 – 1.500 beats/min, their movement being
triggered by reflex mechanisms. Sneezing promotes
the elimination of irritating particles from the nasal
epithelium, and coughing facilitates the expulsion of
bronchial secretions loaded with inert or biological
mycoparticles to the outside, making up the functional
defense mechanisms of the respiratory system [20].
The chemical mechanisms are represented bysubstances with bactericidal effects located on thesurface of the epithelium of the respiratory mucosa(mucus, lysozyme, fibronectin). Mucus provides
additional protection by forming a semipermeablebarrier that ensures the exchange of nutrients, waterand gases, and waterproof at the same time forpathogens [21,22]. It intervenes in the innate immune
defense through mucins, with antimicrobial andantiinflammatory properties [23]. Lysozyme produced
by respiratory epithelial cells, bronchial glands andalveolar macrophages, has bactericidal activity andinfluences the secretion of IgA and complement,
inhibiting the function of neutrophils, by decreasing
chemotactism and oxygen radical synthesis [24].
Cellular fibronectin, secreted along the respiratory
tract by macrophages and fibroblasts, has a role inintracellular adhesion, and plasma fibronectin has arole in opsonization [25]. Fibronectin by competition
blocks the adhesion of bacteria to the cell epitheliumand opposes phagocyte particles, binding to them [26].
The microbiome of the respiratory system isacquired through a dynamic process, which begins atbirth and continues throughout the entire life, showing
major changes with the onset of the chronic lungdisease. The microflora of the respiratory system alsopresents topographic variations [27]. The humanlungs are a low nutrient source for the microorganisms,
compared to the intestinal tract where there is an
exaggerated endogenous microflora [28]. The
phenomena that contribute to the composition of thelung microbiota are immigration of the bacteria (byinhaling bacteria from the inspired air, direct dispersion
along the mucosal surface and microaspiration),
elimination of the bacteria (activity of the mucociliary
apparatus that brings out the microorganisms from thedepth of the lungs) and relative replication rate ofthese germs [29]. The intrapulmonary factors
restricting the development of lung microbiome areblood oxygen, blood flow, local pH, temperature,
Fiziologia - Physiology • 2020.30.2(100)disposition of the effector inflammatory cells and theepithelial cell architecture. Beside these local
physiological factors, the local immune defense
respiratory mechanisms prevent the colonization of
the lung parenchyma [30]. Commensal bacteria are
non-pathogenic and protect the airways against
pathogens through the following mechanisms: they
are the native competitors of the pathogenic bacteria
by tending to occupy the same ecological niche in thehuman body and are able to produce antibacterial
substances called bacteriocins that inhibit the growthof pathogens. The genera Bacillus, Lactobacillus,
Lactococcus, Staphylococcus, Streptococcus andStreptomyces are the main producers of bacteriocins
in the respiratory tract [31]. Chronic lung disease, in
all its clinical forms, besides the architectural changes,
will also cause significant changes in the microbial
ecosystem of the lung [32). Some authors have shownthat in the bronchoalveolar lavage fluid of thesepatients is an abundance of microorganisms
belonging to the phyla Prevotella and Veillonella
genera (from the common anaerobic oral flora), alongwith an increased number of lymphocytes andneutrophils [33]. Subclinical aspiration, the inability ofthe mucociliary apparatus to eliminate this commensal
flora reaching the lower respiratory tract and thedecrease of local immune mechanisms determine thedevelopment of an excessive chronic infection,
characterized by a certain inflammatory phenotype in
BAL (bronchoalveolar lavage) fluid [34]. Therefore, thelung microbiome intervenes in the pathogenesis of thechronic lung disease through its ability to modulate thelocal inflammatory responses and through the
influence of the chronic lung disease [35]. Also, thepatients with NTM lung infections showed a particularinflammatory phenotype on the analysis ofbronchoalveolar lavage fluid, compared to patients
without NTM infections [36]. Therefore, in thesepatients with NTM lung infections, the implications of
the commensal flora reaching the lower respiratory
tract could explain the heterogeneity of the clinicalpicture and the progression of this chronic lungdisease [37].
2.The mechanisms of natural (native or innate)
immune defense include the cells involved in the
inflammatory reaction (neutrophilic, basophilic andeosinophilic polymorphonuclear cells, monocytes,
alveolar macrophages and lymphocytes), NK cells,
cytokines and the febrile process.
Alveolar macrophages, present in a percentage of 90%
in the bronchoalveolar lavage fluid, have mesenchymal
origin and come from the bone marrow, circulating in
the blood as monocytes. In the alveoli, themacrophages may be free or may adhere to thealveolar walls [38]. Macrophages are large cells withmany cellular organs (especially lysosomes andFiziologia - Physiology • 2020.30.2(100)phagosomes), which are the enzymatic basis ofphagocytosis and germ destruction. The complex
enzymatic equipment of these cells contains: proteases,aminopeptidases,
especiallyleucylpeptidase,
esterases,acid
phosphatase,ribonuclease,deoxyribonuclease, lipase, beta-galactosidase, betaglucuronidase and lysozyme. The phagocytic activity ofthe macrophages is nonspecific, representing a barrier
to the microbial or inert particles in the alveoli with thediameter less than 2 mm. At the surface of themembrane, they have receptors for C3 and Fc
receptors for IgG, with a stimulating role inphagocytosis [39]. If the macrophages are transported
on the mucociliary carpet, outside the alveolus, they
become inactive. Neutrophilic polymorphonuclear
cells, present in a proportion of 1% in the
bronchoalveolar lavage fluid, come from the systemic
circulation, adhere to the vascular epithelium bychemotaxis and have effective bactericidal potential
due to a higher density of Fc receptors for the
immunoglobulins on the membrane surface andincreased production of oxygen radicals [40]. The
physiological inflammatory reaction, which occurs
during the non-specific lung defense process, actuallyconsists of: mobilization from the bone marrow of thesecells (neutrophils, monocytes, lymphocytes),chemotaxis, phagocytosis and, finally, intracellulardigestion of microorganisms in the lungs. The effects of
the inflammatory reaction are mediated, in particular, bycomplement and cytokines. The complement system
is a key component in the success of the inflammatory
response, with possible deficiencies leading tosusceptibility to infections. This system consists ofserum proteins (approximately 35), synthesized in
the liver that circulate in inactive form, with enzymatic,
cytolytic and regulatory activity that produce
chemotaxis, opsonization and cytolysis of pathogens.
Activation of the complement triggers the assembly of
the complement's membrane attack complex (MAC).
MAC consists of the complement components C5b, C6,
C7, C8 and C9. The component proteins participate in
the innate immune response both by destroying
microbial agents and by recognizing them, so that they
can be easily identified and destroyed by leukocytes.
MAC increases membrane permeability and triggers
the production of bioactive substances, including
mediators of inflammation (oxygen free radicals) andcytokines. This mechanism has a positive effect on theelimination of pathogens and on the amplification of theinflammatory reaction. Also, the MAC promotes theexpression of p-selectin, which increases the adhesionof neutrophils, platelets and monocytes, then causes
the release of inflammatory mediators [41]. Based on
the practical finding that the incidence of C4 fraction
deficiency is much higher in patients with pulmonary
tuberculosis, it was considered that C4 deficiency could

play a major role in susceptibility to lung NTM infection,
especially in immunocompetent individuals [42]. A
Finnish study showed a statistically significant
difference in serum C4 deficiency in patients with NTM
infections compared to the control group (no serum C4
eficiency) [43]. In addition, the same study showedstatistically significant differences in C4 deficiency,
especially in women with NTM lung infections. It has
been thought that genetic variations of serum
complement will cause a decrease in its ability to attack,
which
makes
nontuberculous
mycobacteria,
possessing a thick lipid layer in the peripheral capsule,become resistant to this physiological defense
mechanism. On the other hand, serum C4 deficiencies
are also associated with certain alleles encoding thesynthesis of HLA histocompatibility antigens. The
frequency of these HLA alleles and haplotypes varies
greatly by population and ethnicity [44,45,46]. The factthat that lung infections with NTM in immunocompetenthosts occur quite frequently in menopausal women inmenopause, it was thought that the aging of theimmune physiological systems could explain thispredisposition to this type of mycobacterial infection.
[47-51] On the other hand, these patients with NTM
lung infections were found to have bronchiectasis much
more often. [52, 53] The findings of this study are that
serum C4 complement deficiency, associated withbronchiectasis in immunocompetent women inmenopause, may be a major risk factor for lung NTM
infections. Cytokines, substances with protein or
glycoprotein structure, synthesized by lymphocytes andmacrophages, act as mediators in the inflammatory
reaction, serving as molecular messengers between
participating cells, with a role in biological amplification.
Lymphokines are soluble mediators, secreted bysensitized lymphocytes, with the role of increasing thephagocytic and enzymatic activity of phagocytic cells on
the opsonized microorganism, as well as against otherintracellular pathogens. NK cells represent 10-20% of
the lymphocyte population and intervene in the innate
immune defense of the host [54], providing protection
against bacteria, viruses and cancer [55-57]. It also
provides "natural" cytotoxicity through the secretion ofcytokines (IFN-γ and TNF) and chemokines (MIP-1α
and MIP-1β), a secretion that supports a proinflammatory environment [58].
NK cells alsointervene in the adaptive immune response, regulate T
cell activity and interact with dendritic cells (DC) [59].
The interaction of NK cells with DC affects the adaptive
immune response and leads to the killing of M.
tuberculosis [60].
The role of NK cells in thepathogenesis of lung infections with NTM wasdemonstrated, initially in mice, in which NK1.1 cell
depletion was shown to exacerbate NTM-induced
pathogenesis by reducing macrophage phagocytosis,
dendritic cell proliferation, cytokine production and
20

granuloma formation. Similar pathological phenomena
have been observed in mice with IFN-γ deficiency after
NTM infection. Also, recombinant IFN-γ infection in
IFN-γ-deficient mice significantly improved immunity
against NTM lung infection. These results indicate that
stimulation of IFN-γ production by NK cells may play animportant role in activating and enhancing innate andadaptive immune responses in the early stages of NTM
lung infection. On the other hand, NK cells stimulate
direct mycobacterial killing and IL-12 production bymacrophages. Fever, as a physiological, generaldefense mechanism of the body has a beneficial role
against infections, because it delays the multiplicationof sensitive microorganisms at high temperatures andaccelerates the metabolism of body cells, while
increasing the immune response and the process ofphagocytosis [61].
3.The specific immune defense mechanisms are
mediated both by the lymphoid tissue cells associatedwith the respiratory mucosa and by the cells of thelymphoreticular system, which intervene in the generaldefense mechanisms (all the immune function cells
that are present in the circulation, lymph nodes andspleen). From a topographical, morphological andimmunological point of view, the respiratory system isdivided into a cranial portion corresponding to thebronchi (where the secretory immune system
predominates) and a distal portion represented byalveoli, where the alveolar macrophages play animportant role in cell-mediated immunity. Bronchusassociated lymphoid tissue (BALT) is composed oflymphoepithelial structures, in which the lymphocytes
come in direct contact with the bronchial andbronchiolar epithelium, participating in the local
pulmonary immune response (secretory IgA formation)[62]. The immunoglobulins, which participate in theanti-infective defense of the lungs, come from the local
and systemic immune compartments. Secretory IgAs
have an antibacterial, antifungal and antiviral role, due
to an agglutination reaction and a reduction in theability of the bacteria to attach to the mucosal surface,
as well as a role in neutralizing toxins [63]. The otherimmunoglobulins (IgM, IgG, IgD, IgE), present in lower
concentrations in bronchial secretions andbronchoalveolar lavage fluid, are involved in: bacterial
agglutination, complement fixation and lysis of gramnegative bacteria (IgM, present only in those with IgAs
deficiency); bacterial agglutination, complement
activation and germ opsonization for the alveolar
macrophages (Ig G); bacterial antigen recognition andantibody synthesis (IgE); increasing bacterial
clearance, by releasing vasoactive amines during theanaphylactic reaction, but also by inhibiting thepenetration of antigen into the mucosa (IgE). Throughthe BALT system, the lung is a unique immune organ,
and the immune events at this level are not necessarily
Fiziologia - Physiology • 2020.30.2(100)reflected in parallel changes in systemic immunity.
Therefore, it is not accurate to draw conclusions about
the normality or abnormality of the pulmonary immune
status, only in base of the serum factors or thecirculating blood cells [64].

CONCLUSION
In conclusion, all pre-existing bronchopulmonary
diseases cause alterations in local immune physiology
and anatomical lesions of the respiratory mucosa,
which explains the predisposition of theimmunocompetent persons to the lung infections

with NTM.
REFERENCES
1.
Francois Portaels,. Epidemiology of Mycobacterial
Diseases. Clin Dermatol; 1995; 13: 207-222.
2.
Pitchenik AE, Fertel D. Tuberculosis andnontuberculous mycobacterial disease. Med Clin North Am
1992; 76: 121-71.
3.
Benson CA, Ellner JJ. Mycobacterium avium complex
infection and AIDS: Advances in theory and practice. Clin
Infect Dis 1993; 17: 7-20.
4.
Portaels F. Le SIDA et les mycobacteries atypiques.
Ann Sot Belg Med Trop 1987; 67: 93-116.
5.
Riello FN, Brigido RTS, Araujo S, et al. Diagnosis of
mycobacterial infections based on acid-fast bacilli test andbacterial growth time and implications on treatment anddisease outcome. BMC Infect Dis 2016; 16: 142-152.
6.
Jeon D. Infection Source and Epidemiology ofNontuberculous Mycobacterial Lung Disease. Tuberc Respir
Dis 2019; 82(2): 94–101.
7.
Swenson C, Zerbe CS, Fennelly S. Host Variability in
NTM Disease: Implications for Research Needs. Front
Immunol 2018; 9: 2901.
8.
Cassidy PM, Hedberg K, Saulson A, Mc Nelly E,
Wintrhrop KL. Nontuberculous mycobacterial diseaseprevalence and risk factors: a changing epidemiology. Clin
Infect Dis. 2009; 49(12): e124-129
9.
Yoo JW, Jo KW, Kang BH, Kim MY, Yoo B, Lee CK,
et al. Mycobacterial diseases developed during anti-tumour
necrosis factor-therapy. Eur Respir J. 2014; 44: 1289-1295.
17. Vareille M, Kieninger E, Edwards MR, et al.The Airway
18. Epithelium: Soldier in the Fight against Respiratory
Viruses. Clin Microbiol Rev 2011; 24(1): 210–229
19. Nicolaescu O, Course V. Pneumonia andsuppurations: Summary. Pathophysiology. Pathogenesis.
Pathological anatomy. Epidemiology. Etiol 2013. Avaible
on www.emcb.ro.
20. Antunes MB, Cohen N. Mucociliary clearance - A
critical upper airway host defense mechanism and methodsof assessment. Curr Opin Allergy and Clin Immunol 2007;
7(1): 5-10.
21. Baraniuk JN, Kim D. Nasonasal reflexes, the nasal
cycle, and sneeze. Curr Allergy Asthma Rep 2007; 7(2): 105111. doi: 10.1007/s11882-007-0007-1.
22. Adler KB, Li Y. Airway epithelium and mucus:
intracellular signaling pathways for gene expression andsecretion. Am J Repsir Cell Mol Biol 2001; 25: 397-400.
23. Fahy JD, Dickey BF. Aitway mucus function anddysfunction. NEJM 2010; 363: 2233-47.
24. Rogan MP, Geragthy P, Greene CM, et al.
Antimicrobial proteins and polypeptides in pulmonary innate
defence. Respir research 2006; 7:29.
Fiziologia - Physiology • 2020.30.2(100)10. Fletcher LA, Chen Y, Whitaker P, Denton M, Peckham
DG, Clifton IJ. Survival of Mycobacterium abscessus
isolated from people with cystic fibrosis in artificiallygenerated aerosols. Eur Respir J 2016; 48: 1789-1791.
11. Griffith DE, Aksamit TR. Understanding
nontuberculousmycobacterial lung disease: it’s been a longtime coming. F1000Res 2016; 5: 2797.
12. Kwon YS, Koh WJ. Diagnosis and treatment of
nontuberculous mycobacterial lung disease. J Korean Med
Sci 2016; 31: 649-659.
13. Stout JE, Koh WJ, Yew WW. Update on pulmonary
disease due to non-tuberculous mycobacteria. Int J Infect
Dis 2016; 45: 123-134.
14.
akansson AP, Orihuela CJ, Bogaert D. Bacterial-HostInteractions: Physiology and Pathophysiology of Respiratory
Infection. Physiol Rev 2018; 98(2): 781–811.
15. Zayas G, Valle JC, Alonso M al. A new paradigm inrespiratory hygiene: modulating respiratory secretions tocontain cough bioaerosol without affecting mucus clearance.
BMC 2007; 7: 11.
16. Labirins NR, Dolovich MB. Pulmonary drug delivery.
Part I: Physiological factors affecting therapeutic
effectiveness of aerosolized medications. Br J Clin
Pharmacol 2003; 56(6): 588–599
25.
oldoveanu B, Otmishi P, Jani P. Inflammatory mechanisms
in the lung. J Inflamm Res 2009; 2: 111
26. Speziale P, Arciola CR, Pietrocola G. Fibronectin and
Its Role in Human Infective Diseases. Cells 2019; 8(12):1516.
27. 26.Vaca DI, Thibau A, Schutz M, et al. Interaction withthe host: the role of fibronectin and extracellular matrix
proteins in the adhesion of Gram-negative bacteria. Med
Microbiol and Immunol 2020; 209: 277-299.
28. Invernizzi R, Lloyd CM, Molyneaux PL. Respiratory
microbiome and epithelial interactions shape immunity in thelungs. Immunol 2020; 160: 171-182.
29. O’Dwyer DN, Dickson RP, Moore BB. The Lung
Microbiome, Immunity and the Pathogenesis of Chronic
Lung Disease. J Immunol 2016; 196(12): 4839–4847.
30. Huffnagle GB, Dickson RP, Lukacs NW. The
respiratory tract microbiome and lung inflammation: a twoway street. Mucosal Immunol, 2017; 10(2): 299-306.

31. Lira-Lucio JA, Falfán-Valencia R,et al: Lung
Microbiome Participation in Local Immune Response
Regulation in Respiratory Diseases. Microorg 2020; 8(7):1059.
32. Availableon:www.en.wikipedia.org/wiki/Lung_microbiota.
33.
Dickson
RP, Huhffnagle GB. The lung microbiome: new principle forrespiratory bacteriology in health and desease. Plos Pathog
2015; 11: e1004923.
34. Segal LN, Clemente JC, Tsay JCT, et al. Enrichmentof the lung microbiome with oral taxa is associated with lunginflammation of a Th17 phenotype. Nat Microbiol 2016; 1:
16031.
35. Lee AL, Goldstein RS. Gastroesophageal reflux
disease in COPD: links and risks. Int J Chron Obstruct
Pulmon Dis 2015; 10: 1935–49.
36. Wnag L, Hao K, Wang C. Role of the Lung
Microbiome in the Pathogenesis of Chronic Obstructive
Pulmonary Disease. Chin Med J (Engl) 2017; 130(17):2107–2111.
37. Kartalija M, Ovrutsky AR, Bryan CL, et al. Patientswith Nontuberculous Mycobacterial Lung Disease Exhibit
Unique Body and Immune Phenotypes. Am J Respir Crit
Care Med 2013; 187(2): 197–205.
38. Johnson MM, Odell JA. Nontuberculous
mycobacterial pulmonary infections. J Thorac Dis 2014;
6(3): 210–220.
39.
Schy
ns J, Bureau F, Marichal T. Lung Interstitial Macrophages:
Past, Present, and Future. J Immunol Res 2018; 5160794.
40. Allen LA, Aderem A. Molecular definition of distinct
cytoskeletal structures involved in complement- and Fc
receptor-mediated phagocytosis in macrophages. J Exp
Med 1996; 184(2):627-37.
41. Wang Y, Jönsson F.Expression, Role, and Regulationof Neutrophil Fcγ Receptors. Front Immunol 2019; 10:1958.
doi: 10.3389/fimmu.2019.01958
42. Markiewski MM, Lambris JD. The role of complement
in inflammatory diseases from behind the scenes into the
spotlight. Am J Pathol, 2007; 171(3):715-27.
43. Swenson C, Zerbe C.S, Fennelly K. Host Variability in
NTM Disease: Implications for Research Needs. Front
Immunol, 2018; 9: 2901.
44. Kotilainen H, Lokki ML, Paakkanen R et al.
Complement C4 deficiency--a plausible risk factor for nontuberculous mycobacteria (NTM) infection in apparently
immunocompetent patients. PLoS One 2014; 9(3):e91450.
45. Senbagavalli P, Kumar N, Kaur G et al. Major
histocompatibility complex class III (C2, C4, factor B)
and C3 gene variants in patients with pulmonary
tuberculosis. Hum Immunol 2011; 72: 173–178.
46. Yuliwulandari R, Sachrowardi Q, Nakajima H, et al.
Association of HLA-A, -B, and -DRB1 with pulmonary
tuberculosis in western Javanese Indonesia. Hum
Immunol 2010; 71: 697–701.
47. Shi GL, Hu XL, Yang L, et al. Association of HLA-DRB
alleles and pulmonary tuberculosis in North Chinesepatients. Genet Mol Res 2010; 10:1331–1336.

22

48. Field SK, Fisher D, Cowie RL. Mycobacterium avium
complex pulmonary disease in patients without HIV
infection. Chest 2004; 126: 566–581
49. Piersimoni C, Scarparo C. Pulmonary infections
associated with nontuberculous mycobacteria in
immunocompetent patients. Lancet Infect Dis 2008; 8: 323–
333.
50. Prince DS, Peterson DD, Steiner RM, et al. Infection
with Mycobacterium avium complex in patients without
predisposing conditions. N Engl J Med 1989; 321: 863–868.
51. Winthrop KL, McNelley E, Kendall B, et al. Pulmonary
non-tuberculous mycobacterial disease. Prevalence andclinical features. An emerging public health disease. Am J
Respir Crit Care Med 2010; 182: 977–982.
52. Han XY, Tarrand JJ, Infante R, et al. Clinicalsignificance and epidemiologic analyses of Mycobacterium
avium and Mycobacterium intracellulare among patients
without AIDS. J Clin Microbiol 2005; 43: 4407–4412.
53. Kim RD, Greenberg DE, Ehrmantraut ME et al.
Pulmonary Nontuberculous Mycobacterial Disease
Prospective Study of a Distinct Preexisting Syndrome. Am J
Respir Crit Care Med 2008; 178: 1066–1074.
54. Chan ED, Iseman MD. Slender, older women appear
to be more susceptible to nontuberculous mycobacteria lungdisease. Gend Med 2010; 7: 5–18.
55. Freud AG, Caligiuri MA. Human naturak killer cell
development. Immunolo. Rev 2006; 214: 56-72.
56. Smyth MJ, Hayakava Y, Takeda K et al. New
aspects of natural-killer-cell surveillance and therapy ofcancer. Nat Rev Cancer 2002; 2: 850-861.
57. Vivier E, Tomasello E, Baratin M et al. Functions of
natural killer cells. Nat Immunol 2008; 9: 503-510.
58. Bjorkstrom N, Ljunggren H.G, Michaelson J.
Emerging insights into natural killer cells in human periferal
tissues. Nat Rev Immunol 2016; 16: 310-320.
59. Huntington ND, Vosshenrich CA, Di Santi JP.
Development pathways that generate natural-killer-celldiversity in mice and humans. Nat Rev Immunol 2007; 7:
703-714.
60. Raulet D. Interplay of natural killer cells and their
receptors with the adaptative immune response. Nat
Immunol 2004; 5: 996-1002.
61. Schierloh P, Aleman M, Yokobori N et al. Nk cell
activity in tuberculosis is associated with impaired CD 1a,and ICAM-1 expression: a regulatory role of monocytes in
NK activation. Immunology 2005; 116: 541-552.
62. Plaza JJG, Hulak N , Zhumadilov Z et al. Fever as animportant resource for infectious diseases research.
Intractable Rare Dis Res 2016; 5(2):97-102.
63. Randall TD. Bronchus-associated lymphoid tissue(BALT) structure and function. Adv Immunol. 2010; 107:187241.
64. Corthésy B. Multi-faceted functions of secretory IgA at
mucosal surfaces. Front Immunol 2013; 4:185.
65. Pabst R, Gehrke I. Is the Bronchus-associatedLymphoid Tissue (BALT) an Integral Structure of the Lung in
Normal Mammals, Including Humans? AJRCMB 1990;
3(2):131-5.

Fiziologia - Physiology • 2020.30.2(100)SEMNIFICATIA ALTERARII MECANISMELOR DE
APARAREANATOMICESIFIZIOLOGICERESPIRATORIIINPATOGENEZA
BOLII
PULMONARE DATORATE MICOBACTERIILORNONTUBERCULOASELAGAZDELEIMUNOCOMPETENTEREZUMATIn prezent, boala pulmonara datorata NTM (BP-NTM) este o problemă de sanatate publica datorita cresterii
proportiei în comunitate a gazdelor umane imunocompromise, pe de o parte prin cresterea incidentei infectiei HIVSIDA, dar mai ales prin folosirea pe scara larga a chimioterapicelor antineoplazice si a anticorpilor monoclonali(anti-TNF si anti-IFN). Ca urmare, costurile cu diagnosticarea si spitalizarea acestor pacienti vor fi din ce in ce maimari, punandu-se o presiune financiara asupra sistemelor de sanatate publica din tarile dezvoltate, dar mai ales,
din cele aflate in curs de dezvoltare. In prezent, un deziderat important al medicilor este diagnosticarea precocesi corecta a acestor infectii pulmonare, cu atat mai mult cu cat micobacteriile nontuberculoase sunt omniprezente
si pot contamina orice probă de produs biologic recoltat de la bolnav.
Cuvinte cheie: aparare a sistemului respirator, NTM, gazde imunocompetente.

Fiziologia - Physiology • 2020.30.2(100)23

ASSESSMENT OF SERUM LEVELS OF THE ANTIINFLAMMATORY CYTOKINE IL13 IN OBESE AND
TYPE 2 DIABETES PATIENTSIOANA DANIELA BINIȘOR1, RALUCA ARGENTINA MOLDOVAN2,IONUȚ MOLDOVAN2, RAREȘ DIN ANGHEL3, CĂTĂLINA GABRIELAPISOSCHI3, ILEANA MONICA BANIȚĂ3
1Medical Center Binisan, Drăgășani, Romania
2Emergency County Hospital Alba Iulia, Romania
3University of Medicine and Pharmacy Craiova

ABSTRACT
Aim: To study the circulating levels of IL13 in obese and diabetic patients. Methods: The study group included 69
patients from an outpatient clinic, divided in four subgroups: obese without diabetes (O) (n= 24), obese with diabetes(OD) (n= 24), non obese diabetics (NOD) (n= 9) and controls, lean individuals without diabetes (C) (n= 12). Several
clinical (age, sex, weight, BMI, abdominal and hip circumference, blood pressure), laboratory (blood glucose,glycated hemoglobin, serum proteins and protein electrophoresis, lipid metabolism parameters, urea and creatinine,
CRP and IL13 serum levels) were assessed in the study group and separately in each subgroup. Statistical analysis
was performed using the appropriate statistic tests. Results: IL13 was significantly higher in the OD versus the Cgroup (27.61±9.72 pg/ml vs 20.69±5.89 pg/ml, p= 0.03). There was a moderate positive correlation between IL13
and abdominal circumference (correlation coefficient rs 0.416, p=0.0004). In multivariate regression analysis,
abdominal circumference and IL4 were significant predictors of IL13 (regression model p=0.0086). No statisticallysignificant differences in IL13 levels were found between the NOD and the control groups. Conclusions: Abdominal
obesity was associated with higher levels of IL13 which was negatively correlated with C-reactive protein level.
Diabetes in non obese individuals was not associated with alterations in the IL13 levels.
Keywords: serum level IL13, anti-inflammatory cytokines, obesity, diabetes

INTRODUCTIONWhite adipose tissue (WAT) is composed of
adipocytes, as well as of a non-adipocitary stromovascular fraction that includes undifferentiated cells,
preadipocytes, fibroblasts, inflammatory cells andvarious amounts of vessels and nerves; adipocytes
and pro-inflammatory resident immune cells
populations,primarily
macrophages
andlymphocytes. These cells secrete several pro-and
antiinflammatory cytokines, called adipokines,
accounting for the main part of the so called adipose
secretoma (43). These molecules are implicated invariousphysiologicalprocesses:
energyconsumption
andstorage,temperatureaccommodation and in immunity.
In the obese subjects, the adipose tissue is not only
overdeveloped, but the immune cells inside thestromo-vascular fraction also rise in number andchange in phenotype. Consequently, the secretion ofsome adipokines is deregulated, mainly in thevisceral location of adipose tissue, where apparently
the infiltration of macrophages is more important (19,39, 13); a chronic low intensity inflammation status isestablished. Although subclinical, this mild
inflammation, defined as metabolic inflammation (25)is associated with important systemic consequencessuch as type 2 diabetes mellitus, (DM) metabolic
syndrome, (Mets) cardiovascular diseases andcertain types of cancer (35, 2, 17, 29, 15). It has also
been shown that the dysfunction of the subcutaneous
adipose tissue is involved in the pathogenesis ofMets (7).
The pro-inflammatory pathway in Mets is notably
induced by lymphocyte T helper Th1-Th2 polarization
with consequent derived cytokine synthesis. Th1

Received 30th of August, 2020. Accepted 25th of September, 2020. Address for correspondence: Rares Din Anghel, Universityof Medicine and Pharmacy of Craiova, Romania; Craiova, 2 Petru Rares St., Romania; phone: 0757834539; email:raresdinanghel@yahoo.ro

24

Fiziologia - Physiology • 2020.30.2(100)cytokines(IL-12, IL-2 and interferon gamma (IFN-γ)) determine
the pro-inflammatory M1 phenotype of macrophages.
Macrophages undergoing the alternative activation
by Th2 cytokines (IL-4 and IL-13), M2a macrophages(26) trigger a different immune response (28). The
Arginase-1 gene expression is the signature of thealternative activation (23, 28, 42).
These two phenotypes of macrophages have been
well studied in obese mice: the majority of themacrophage population consists of the M1 cells,
characterized by high levels of pro-inflammatory
cytokines, such as TNF-alpha, production of INOS
(inducible nitric oxide synthase) and oxygen reactive
species and by a strong microbicidal and tumoricidal
activity (40). In the lean mice, most of themacrophages, with the M2 phenotype, arecharacterized by the expression of genes encoding
anti-inflammatory proteins such as arginase -1, IL-10,
considered to be involved in parasite immunity, tissue
remodeling and tumor progression (6, 23, 40).
The source of the IL-13 cytokines in the adipose
tissue is still under debate. There are discrepancies
between the in vitro and in vivo studies, as well as
between the animal and human modelsof alternative macrophage activation.
In mice, the source of IL-13 are the eosinophils that
accumulate in the adipose tissue: their number in the
perigonadal adipose tissue positively correlated withthe arginase 1-positive macrophages (45). The
adipocytes themselves (18) could be a source of IL13. In mice, exogenous administration of otherinterleukins (IL-25 and IL-33) leads to a marked riseof the IL-4 and IL-13 levels (38).
In animal models, the paradigm of Th1/Th2 cytokines
triggering the M1/M2 activation seems to be the clueof the onset and progression of inflammation in themetabolic syndrome.
Few studies are available on circulating IL-13 inobese individuals (15, 32) and very few papers exist
on the relationship between these cytokines anddiabetes (24). In a previous study, we showed theincrease of IL-4 levels in obese and obese diabetic
patients and its correlation with biochemicalparameters (5).

AIMS

In the current study, we aim to determine therelationship between IL-13 plasma levels andadipometric and metabolic parameters in obese andobese diabetic human subjects, and to check thecorrelation between IL13 and IL4.

Fiziologia - Physiology • 2020.30.2(100)

MATERIAL AND METHODS:
Subjects. The study included 69 subjects with complete
anthropometric, adipometric and biochemical
examinations recruited from the patients of theoutpatient clinic Binisan Medical Center, Dragasani.
Patients presenting with medical emergencies, acuteand chronic infections, parasitosis, malignancies andthose treated with drugs impacting the immune systemwere excluded. Informed consent was obtained fromevery participant and the study was approved by the
Ethics Committee of the University of Medicine andPharmacy from Craiova, Romania.
Subjects were divided into four subgroups:- the O subgroup (overweight and obese), n =24, including overweight or obese subjects (body massindex (BMI) ≥25) without diabetes. Six obese or
overweight individuals previously not known asdiabetics and discovered with diabetes during the study
were further assigned to the OD group;- the OD subgroup (overweight and obese
diabetics), n = 24, patients with diabetes and a BMI ≥25;
- the NOD subgroup (non-obese diabetics), n =9 lean individuals, known with diabetes and a BMI <25;
- the C subgroup (controls), n=12, with a normal
body weight (BMI<25) and without diabetes.
Anthropometric and adipometric measurements. Sex,age, height, body weight, waist and hip circumference,systolic and diastolic blood pressure, body mass index
were registered.
Height (cm) was measured using a stadiometer andbody weight (kg) was recorded with a calibrated digitalscale with subjects in underwear. Waist circumference(WC, cm) was measured at the midpoint between thelast rib and the upper point of the iliac crest. Hip
circumference (HC, cm) measurement was taken at thewidest lateral extension of the hips. Blood pressure wasmeasured after 5 minutes rest using a
sphygmomanometer before venipuncture for blood
sample collection. BMI (kg/m2) was defined as bodyweight divided by the square of height.
Biochemical measurements.
Blood samples were collected after an overnight fast,
early in the morning. For each subject, threevacutainers were drawn: two without anticoagulant, withseparating gel, to obtain serum samples for biochemicalparameters detemination and one with K3EDTA in order
to measure hba1c level.
After blood clotting, for at least 30 minutes, thevacutainers were centrifuged at 1000 rpm for 10
minutes and serum was immediately aliquoted inpolypropylene tubes.
After aliquoting, samples were either processed forserum biochemistry or stored at -80oc for cytokine
measurement.

25

Assay), Biolegend, kit lot B194484 dedicated to the
Personal LAB CLIA Fully Automated 2 Microplate
Analyser CLIA\ELISA (Adaltis).
Statistical analysis
Clinical and the above mentioned biochemicalparameters were registered and analysed for the entire
study group and separately for each of the four
subgroups.
Numeric variables were expressed as averages ±standard deviations for the normally distributed ones
and as median (25-75 percentiles) for the non-normally
distributed ones. Qualitative variables were expressedas percentage. Numeric variables were comparedusing the t test for normal distribution and the Mann
Whitney test for non-parametric variables. Qualitative
variables were compared using the Chi-squared test.
Correlation between parameters was studied using thePearson correlation coefficient for parametric variables
and the Spearman correlation coefficient for the nonparametric variables. Univariate linear regression andmultivariate regression were used to investigate therelationship between IL-13 and the clinical andbiochemical parameters.
The statistical significance threshold chosen was
p<0.05. The statistical analysis was performed using
the Microsoft Excell software and the medcalc v.16.1
software.

For each patient, two vacutainers without additives, withseparator gel to obtain serum and one K3 EDTA
vacutainer were collected.
After aliquoting, samples were processed according toeach particular analysis, and tested using the following
analytical methods:
For the serum assesment of biochemical parameters,
the methods used were:
GOD PAP method for glucose, the CHOD PAP method
for cholesterol, the PAP GPO method for triglycerides,
direct methods for HDL, the Biuret method without blanksample for total protein, the Jaffe method withoutcompensation for creatinine, the Urea method -UV for
urea, glycated hemoglobin (hba1c) by direct method, Creactive protein
by Fixed time method; serum
proteins electrophoresis by the method of migration in
agarose gel in the electric field; the Jaffe method
without compensation for creatinine was processed.
LDL-cholesterol was calculated by the Friedewald
formula. Glycated hemoglobin (hba1c) by direct method,using a Biotechnica kit, lot 512503.
High-sensitivity CRP values were determinedturbidimetrically using fixed-time measurement withsample blank correction using a CRP FUTURA Kit, lot
0299-2299 02102-22102 EXP 11.2016.
All Biochemical measurements were performed with
Biotechnica kits, using an automated analyzer BT3500.
The method used for IL13 concentration mesurement
was ELISA (Sandwich Enzyme-Linked Immunosorbent

Results:

The results are depicted in table 1
Table 1.
Study parameters in the O, OD, NOD and M groups and statistical significance p betwen study
subgroups and the control group.
Parameters

Group O
N=24

Group OD
N=24

Group
NOD
N=9

Group C
N=12

p1

p2

p3

Age(years)

57.41±11.94

63 (58-66.5)

61.44±9.9828.6±5.5

0.011<0.0001<0.00
01

Sex(M/total)

13/24

13/24

4/9

10/12

0.18

0.18

0.16

Weight(kg)93.5(79.5-104.5)

87.45±11.73

62(59.7571)

69.33±7.26

0.0001<0.0001

0.28

Height(cm)170(164.5-177.5)

167.2±9.05

169(166.2
5-175)

175.91±5.93

0.064

0.00480.26

BMI(kg/m2)

29.54(27.3-34.36)

31.24±3.07

22.42±1.4722.36±1.53<0.0001<0.0001

0.93

Abdominal
circumference(cm)

108.08±15.02

112.33±8.58

88.11±8.4

82.83±7.01<0.0001<0.0001

0.13

CLINICAL

26

Fiziologia - Physiology • 2020.30.2(100)Parameters

Group O
N=24

Group OD
N=24

Group
NOD
N=9
99±6.06

Group C
N=12

p1

p2

p3

Hip
circumference(c
m)

115±9

115.5(111-122)

100.91±5.05<0.0001<0.0001

0.43

Systolic bloodpressure(mmHg)

132.2±17.21

141.41±17.15

137.44±22.22117.83±11.2

0.013

0.0001

0.015

Diastolic bloodpressure(mmHg)

78.5(70-86.5)

79.3±8.7880(8092.5)

68.83±10.32

0.4

0.00320.003

7.54(7.14-7.75)

7.69±0.56

7.74±0.41

7.87±0.43

0.018

0.35

0.51

Albumin%

64.9(63.05-67)

64.98±3.63

63.78±3.9967.83±4.08

0.04

0.042

0.04

Alpha1%2.2±0.31

2.43±0.46

2.67±0.59

2.17±0.63

0.12

0.18

0.09

Alpha2%9.72±1.1

9.95±1.2

10.73±1.198.78±1.49

0.04

0.017

0.006
4

Beta1%

7.45±0.83

7.85±1.14

7.91±1.07

6.5±0.62

0.00150.00070.001
5Beta2%

3.5(3.25-3.8)

3.71±0.89

3.48±0.59

3.15±0.81

0.16

0.08

0.34

Gamma%

11.45(10.4-13.75

11.05±2.35

11.4±2.35

11.54±2.53

0.82

0.57

0.9

Albumin (g/dl)

4.87(4.64-4.97)

4.95±0.42

5.21±0.79

5.33±0.41

0.002

0.1

0.07

Alpha1 (g/dl)

0.18(0.16-0.21)

0.18±0.03

0.21(0.19-1.26)

0.17±0.04

0.48

0.4

0.058

Alpha2 (g/dl)

0.73±0.08

0.76±0.1

0.82±0.12

0.68±0.1

0.17

0.035

0.013

Beta1 (g/dl)

0.56±0.07

0.61±0.08

0.59±0.08

0.51±0.06

0.07

0.00250.019

Beta2 (g/dl)

0.27±0.05

0.28±0.07

0.26±0.04

0.25±0.07

0.33

0.23

0.57

Gamma (g/dl)

0.87(0.79-1.04)

0.84±0.21

0.88±0.18

0.94±0.25

0.81

0.24

0.56

Serum Glucose(mg/dl)95(86.5-108)

150.95±40.29

155.77±63.5289.41±6.05

0.057<0.0001

0.001
8

HbA1C%

5.78±0.54

7.2(6.65-8)

6.67±1.24

5 (4.5-5.1)

0.0007<0.00010.000
8656.09±124.3
1678.049±132.72

718.25±14
5.92564.42±127.
4

0.05

0.019

0.018

LDL (mg/dl)

136.83±46.27

128.02±49.33

141.4±36.
53

115.62±43.8
3

0.19

0.46

0.16

HDL (mg/dl)

44.92(38.12-51.57)

43.17(39.43-48.13)

40.99±6.3245.25±14.27

0.86

0.81

0.41

PARACLINICAL
Total(g/dl)

Total(mg/dl)Protein

LipidsFiziologia - Physiology • 2020.30.2(100)27

Parameters

Group O
N=24

Group OD
N=24

Group
NOD
N=9
30.04±16.
17Group C
N=12

p1

p2

p3

VLDL (mg/dl)

18.62(15.34-23.1)

29.45±9.78

15.82±6.29

0.16

0.0001

0.013

Total Cholesterol(mg/dl)203.64±50.1

202.46±51.55

212.44±40.59176.79±46.2
4

0.12

0.15

0.08

Triglycerides(mg/dl)87.92(73.95-113.96)

147.29±48.94

150.24±83.8979.13±31.44

0.22

0.00010.013

IL4 (pg/ml)

0.49(0.1-2.4)

4.72±3.8

0.51(0.1-1.44)

0.3±0.3

0.075

0.0003

0.28

IL13 (pg/ml)

24.18±6.33

27.61±9.72

22.63±6.0
920.69±5.89

0.12

0.03

0.47

CRP (mg/dl)

2.1(0.83-2.82)

2.75(1.39-4.68)

0.19(0.1-3.39)

1.68±1.85

0.26

0.05060.93

Urea (mg/dl)

34.13(30.32-40.63)

36.38±11.56

34.6(27.2648.27)

30.81±7.57

0.056

0.14

0.35

Creatinine(mg/dl)0.86±0.15

0.88±0.21

0.75(0.69-0.76)

0.96±0.12

0.65

0.28

0.075

Creatinine
clearance(ml/min)

118.62±44.13

102.81±19.82

87.27±12.
08111.4±11.14

0.26

0.96

0.1

Legend –Table 1.
Study parameters were expressed as mean ± standard deviation for normal distribution and as median(25-75 percentile) for abnormal distribution.
p1 refers to the comparisons between groups O and M, p2 refers to the comparisons between groups
OD and M, p3 refers to the comparisons between groups NOD and M. Statistical tests used were t-test
for normally distributed variables and the Mann Whitney test for abnormal distribution. Comparison of
proportions was performed using the chi-squared test.
The assesment of IL4 values was communicated in a previous work (5) but we considered the values
for comparison (Tables 2, 3).
Table 2. Corelation of IL13 with study groupparameters

28

Parameter

Correlation
coefficient
(rS)

P

Age

0.279

0.0204

Abdominal
circumference

0.416

0.0004Hip circumference

0.311

0.0092

Weight0.206

0.0894IL4

0.401

0.0006

Fiziologia - Physiology • 2020.30.2(100)There was a moderate positive correlation between IL13 and IL4 and abdominal circumference and a weakcorrelation of IL13 with age and weight.
Table 3. Simple regression with IL13 as a dependent variable
Parameter

Determination coefficient

P

Age

0.08

0.02

Abdominal circumference

0.17

0.0004Hip circumference

0.1

0.0092

Weight0.04

0.0894IL4

0.16

0.0006

Fiziologia - Physiology • 2020.30.2(100)29

In multivariate regression analysis, abdominal
circumference and IL4 remained significant predictors of
IL13 (p=0.0086).The patients in the control group weresignificantly younger than those in the other groups.
As expected, in the O and OD groups, there werehigher BMI, abdominal circumference and hip
circumference . The abdominal circumference wassignificantly higher in the OD group versus the O group(p=0.0047), thus the OD patients were more obese thanthe ones in the O group. There was no significant
difference between these parameters in the O versus
the OD group. Systolic blood pressure was higher in all
the study groups than in the control group. Diastolicblood pressure was higher in diabetic patients, in obese
as well as in non obese patients.
Differences were found in the biochemical parameters
as follows: the obese patients (O group) had a poorer
nutritional status, with lower total protein and lower
albumin.
There were signs of inflammation in the obese group, as
well as in the obese and non-obese diabetics, withhigher Alpha 2 and Beta 1 globulins.
As expected, the blood sugar was higher in the diabetic
groups, but there was a tendency of higher blood sugarin the obese group without diabetes than in the control
group. Hb A1c was significantly higher in the diabetic
groups, but also, significantly higher in the obese
compared to the control group.
As for the lipid profile, significantly higher VLDL andtriglyceride levels were found in both OD and NOD
groups versus the control group.
CRP was modestly elevated (tendency) in the OD group.
The inflammatory molecules IL4 and IL13 weresignificantly higher in the OD versus the C group.
Significant correlations with IL13 were found for: age,
abdominal and hip circumference, weight and IL4 (Table
2).
In univariate regression analysis, significant association
was found for the same parameters. In multivariate
regression analysis, with IL13 the dependent variable,
significant predictors of IL 13 remained abdominal
circumference and IL4 (regression model p=0.0086).

DISCUSSION:
The macrophage of the adipose tissue could beconsidered one of the most important actors, film
director and screenwriter of metaiflammation in obesity,
acting by the secretion of different type of chemokines.
In the present study we performed an IL-13 serum levelassessment for 69 subjects divided in four groups as we
previously described, and we compared the values
found with some anthropomorphic, adipometric andbiochemical measurements. A statistical correlation was
also made, also taking into account the results regarding
30

the IL-4 serum values published in a previous work insame groups of patients (5).
The WAT depots in obesity suffer an important plasticity
and remodelling in term of adipocyte population and theinnate and secondary immune cells phenotype. A lot of
research emphasize the increased number ofmacrophages in the stromovascular fraction of obese
subjects the change of their location in adipose lobules
among the adipocytes and, more important, thedramatically change of their phenotype and their effecton adipogenesis and glucose uptake (13, 7, 23, 6, 10,
16, 44).
IL4 and IL13 are glucocorticoid hormones acting as
immunomodulatory cytokines, produced mainly in leansubjects by adipocytes and activated Th2 cells and are
inducers for M2a polarization of macrophages (26).
Studies in animal models and cell cultures proved thatbreaking the M2 activation leads to obesity and insulin
resistance (22, 31). IL4 and IL13 are also involved inmaintaining T regulator cells and eosinophils number(26, 28). The gene encoding IL13 is part of a cluster ofgenes on chromosome 5q, particularly close to IL4 (14).
The simultaneous variation of Il-13 and IL-4 as we also
have demonstrated is probably linked to the vicinity of
the genes encoding the two cytokines on the 5q
chromosome, probably responding to similar activation
pathways (4).
The sequence of events in the activation of immune
cells in the adipose tissue under hypercaloric intake
seems to be important for understanding thepolarization of macrophages and their response toinflammation. The Th1 cytokines activity, leading to theM1 macrophages activation by the so-called classicalway, responsible for the metainflammation in mets, isvery well documented. Makki et al (25) showed thatthe alterations in macrophage content and polarisation
state (M2-M1) occur fairy late in the progression ofobesity and they are not the initiating events ofinflammation. The adipose tissue of lean subjects
secretesanti-inflammatory
cytokines:
TGFβ,
adiponectine and IL10, IL4 and IL13 (25). Macrophages
are the late effectors of a coordinated inflammatory
response, where the initial occurrences are theaccumulation of proinflammatory T cells, (CD8+ andTh1CD4+), depletion of regulatory T cells, accumulationof B cells, NK cells, eosinophils, neutrophils, and mast
cells. The resulting M1 macrophages produce
proinflammatory cytokines such as IFN-γ and TNF–α(37). Consecutively, the low-grade inflammation
installed in the obese adipose tissue is followed by Mets,
insulin resistance, arterial hypertension and asthma (37).
The Th1 cells also play a critical role in the initiation,
progression and rupture of atherosclerotic plaque (3).
The role of the Th2 antiinflammatory cytokines IL4 andIL13 is poorly understood and controversial and the
Fiziologia - Physiology • 2020.30.2(100)interest in the IL13 involvement in human obesity is
recent. The alternative activation of M2 macrophages
by IL4 and IL13 is involved in the type II inflammation
processes, like allergy and killing and encapsulation ofparasites (26). In mice, the M2 macrophage
differentiation seems to be triggered by thehypercaloric diet and the initial proinflammatory statein early obesity aims to improve the insulin sensitivity(46).
Il 13 down-regulates the activity of the macrophages,
inhibiting the production of inflammatory cytokines (23,25). Higher serum concentrations of IL13 have been
associated with reduced weight gain in rodents thatwere fed a high fat diet and they limited diet induced
inflammation and insulin resistance (11, 21).
In humans, the association between IL13 on one handand obesity and DM on the other hand is poorly
understood. The present paper is one of the few
studies to investigate the relationship between type 2
diabetes and anti-inflammatory cytokines. Our results
confirm the findings of previous studies (3, 9, 42). Ourfindings add data to the body of evidence of increased
Th2 cytokine serum levels in obese individuals. This
could be expected, considering the cytokine secreting
cells accumulation and adipose tissue expansion inobese. The patients in the OD group had higher IL13
serum levels, positively. As displayed in table 1, theobesity was more severe in the OD group than in theother study groups.
Schmidt and coworkers (37) showed that both pro-and
anti-inflammatory cytokines are elevated in obese
subjects and pointed out the positive correlation
between physical activity and cytokine levels.
Surendar et al demonstrated that both Th1 and Th 2
cytokines are upregulated in Mets (42).
Similar results (1) reported an increase in serum levelsof Il-4 and Il-13 in diabetic subjects. They noted thatthe diabetic nephropathy is characterised by a declineof Il-33, the master regulatory of Th-2 differentiation,
meanwhile the subjects with uncomplicated DM
maintain elevated levels of Il-13. The authors suggestthat Th2 cytokines may have a protective effectagainst the complications of diabetes mellitus.
The high levels of antiinflammatory cytokines in theobese individuals are probably adaptive, reducing theproinflammatory effect of several other cytokines
originating from the resident immune cells in the
adipose tissue.
Effects such as insulin resistance reduction in obesity
have already been demonstrated (21). Whether
different levels of IL-13 and IL-4 in obese humans
would be able to stratify or influence the insulin
resistance and the diabetes risk in these patientsremains to be established. There are animal studies
showing that IL4 promotes glucose tolerance, inhibits
Fiziologia - Physiology • 2020.30.2(100)adipogenesis and promotes lipolysis in matureadipocytes (21).
In vitro studies have shown cytoprotective effects ofantiinflammatory cytokines I13 and IL4 on thepancreatic beta cells (27, 36, 41).
In multivariate regression analysis, diabetes control(HbA1c) was not a significant predictor of IL-13 levels.
Moreover, IL-13 and IL-4 levels were not different
between the non obese diabetic group and controls,raising the hypothesis that higher IL-13 and IL-4 levelsin obese diabetic patients are probably linked toobesity and not diabetes.
The fact that our study failed to show a link between
diabetes control and IL13 levels may be due to the factthat diabetes was under treatment. The interleukin
levels have recently been demonstrated to be reducedin patients treated with sulfonylureas, in contrast withpatients on insulin, who exhibit higher inflammatory
activity (20). For the present study, data on treatment
was not collected.
Several clinical effects of Il l3 have been studied. It was
shown to increase asthma activity and an IL13 inhibitor
has been proposed in the treatment of uncontrolled
asthma (34).
An important clinical and biochemical observation isthe existence of metabolically healthy obese subjects,
who do not suffer from the complications of obesity,
displaying instead high serum levels of antiinflammatory Th2 cytokines. They potentiallyrepresent a variant obese phenotype (12), but one
must consider the age of these subjects, their genetic
background and the physical activity.
An elegant experiment of Nguyen (30) demonstrated
that in mice exposed to cold, the secretion of IL-4
stimulates the alternatively activated macrophages M2
in adaptative thermogenesis, both in white and brown
adipose tissue. As a result, there is an increase inuncoupling respiration and energy expenditure bynoradrenaline (8). Animal models have shown that
adaptative thermogenesis acts as a protective
mechanism against high fat diet induced obesity. The
alternatively activated M2 macrophages secreting
catecholamines could be a very important factor in thebrowning process of a subset of cells located in WAT
depots, that could be switched into brown-likeadipocytes, named “beige” or “bright” (44).
In agreement with the supposition that IL13 has aprotective effect in obesity, our study showed a very
weak correlation between the acute phase-protein
CRP, IL-4 and IL-13 in the obese and obese diabetic
group. CRP is one of the most important proinflammatory markers in atherosclerosis and type 2
DM, synthesized in response to proinflammatory
cytokines such as TNF α and IL-1 (33).
Our results corroborated with the few data available

31

from similar research in humans suggest theprotective anti-inflammatory role of IL13 cytokine in theobese diabetic and their involvement in preserving themetabolic status of these patients.

CONCLUSIONS:

Obesity is associated with higher levels ofantiinflammatory cytokine IL-13. IL-4 and IL- 13

Acknowledgements:
This work is a result of a research made possible by

REFERENCES
1. Anand G, Vasanthakumar R, Mohan V, et al: Increased IL12 and Decreased IL-33 Serum Levels Are Associated with
Increased Th1 and Suppressed Th2 Cytokine Profile inPatients with Diabetic Nephropathy (CURES-134). Int J Clin
Exp Pathol 2014;7(11):8008-8015.
2. Antuna-Puente B, Feve B, Fellahi S, et al: Adipokines: themissing link between insulin resistance and obesity.
Diabetes Metab 2008;34(1):2-11.
3. Aravindhan V, Madhumitha H: Metainflammation in Diabetic
Coronary Artery Disease: Emerging Role of Innate andAdaptive Immune Responses. J Diabetes Res 2016;
6264149.
4. Bhattacharjee A, Shukla M, Yakubenko VP et al: Il-4 and Il13 employ discrete signaling pathways for target gene
expression
inalternatively
activatedmonocytes/macrophages. Free Radic Biol Med 2013;54:116.
5. Binisor ID, Moldovan R, Moldovan I et al: Abdominal Obesity
and Type 2 Diabetes Mellitus are Associated With Higher
Seric Levels of IL 4 in Adults. Curr Health Sci J
2016;42(3):231-237.
6. Boutens L, Stienstra R: Adipose tissue macrophages: going
off track during obesity. Diabetologia 2016; 59(5):879-894.
7. Bremer AA, Jialal I: Adipose tissue dysfunction in nascentmetabolic syndrome. J Obes 2013;393192.
8. Cannon B, Nedergaard J: Nonshivering thermogenesis andits adequate measurement in metabolic studies. J Exp Biol
2011;214:242-253.
9. Chen H, Wen F, Zhang X et al: Expression of T-helperassociated cytokines in patients with type 2 diabetes mellitus
with retinopathy. Mol Vis 2012;18:219-226.
10. Cinti S, Mitchell G, Barbatelli G et al: Adipocyte death
defines macrophage localization and function in adipose
tissue of obese mice and humans. J Lipid Res
2005;46(11):2347-2355.
11. Darkhal P, Gao M, Ma Y et al: Blocking high-fat diet-induced
obesity, insulin resistance and fatty liver by overexpressionof Il-13 gene in mice. Int J Obes (Lond). 2015;39(8):12921299.
12. Denis GV, Obin MS: Metabolically healthy obesity: origins
and implications. Mol Aspects Med 2013;34(1):59–70.
32

display similar values in obesity and diabetes.
Diabetes was not associated in our study withalterations in IL-13 serum levels.
Further studies are necessary to understand theimplication of Th2 cells in all the secondary diseasesof human obesity and to link the effect to the cause,checking the phenotype of macrophages in adipose
depots in both general and central obesity.

the support of MD Codrin Bernevig and Biologist
Andreea Simion from Proton Life Science

13. Fusaru V, Taisescu C, Bold A et al: Role of innate immune
receptors TLR2 and TLR4 as mediators of the inflammatory
reaction in human visceral adipose tissue. Rom J Morphol
Embryol 2012;53(3 Suppl):693-701.
14. Gray KA, Yates B, Seal RL et al: Genenames.org: the HGNC
resources in 2015. Nucleic Acids Res 2015;43: D10791085.
15. Heilbronn LK, Campbell LV: Adipose tissue macrophages,
low grade inflammation and insulin resistance in humanobesity. Curr Pharm Des 2008;4:1225–1230.
16. Hotamisligil GS, Arner P, Caro JF, et al: Increased adipose
tissue expression of tumor necrosis factor-alpha in humanobesity and insulin resistance. J Clin Invest
1995;95(5):2409-2415.
17. Kahn BB, Flier JS: Obesity and insulin resistance. J Clin
Invest 2000; 106(4):473-481.
18. Kang K, Reilly SM, Karabacak V, et al: Adipocyte-derived
Th2 cytokines and myeloid PPARdelta regulate macrophage
polarization and insulin sensitivity. Cell Metab
2008;7(6):485-495.
19. Kissebah AH, Vydelingum N, Murray R, et al: Relation ofbody fat distribution to metabolic complications of obesity. J
Clin Endocrinol Metab 1982;54(2):254-260.
20. Kothari V, Galdo JA, Mathews ST: Hypoglycemic agentsand potential anti-inflammatory activity. J Inflamm Res
2016; 9:27-38.
21. Kwon H, Laurent S, Tang Y, et al: Adipocyte-Specific IKKβSignaling Suppresses Adipose Tissue Inflammation throughan IL-13-Dependent Paracrine Feedback Pathway. Cell Rep
2014;9(5):1574-1583.
22. Liao X, Sharma N, Kapadia F, et al: Krüppel-like factor 4
regulates macrophage polarization.J Clin Invest
2011;121(7): 2736-2749.
23. Lumeng CN, Del Proposto JB, Westcott DJ, et al:
Phenotypic switching of adipose tissue macrophages withobesity is generated by spatiotemporal differences inmacrophage subtypes. Diabetes 2008;57(12):3239-3246.
24. Maier R, Weger M, Haller-Schober EM, et al: Multiplex beadanalysis of vitreous and serum concentrations ofinflammatory and proangiogenic factors in diabetic patients.
Mol Vis 2008;14:637-643.
25. Makki K, Froguel P, Wolowczuk I: Adipose tissue in obesityrelated inflammation and insulin resistance: cells, cytokines,
and chemokines. ISRN Inflamm 2013;139239.
Fiziologia - Physiology • 2020.30.2(100)26. Mantovani A, Sica A, Sozzani S, et al: The chemokine
system in diverse forms of macrophage activation andpolarization. Trends Immunol 2004;25(12):677-686.
27. Marselli L, Dotta F, Piro S, et al. Th2 cytokines have apartial, direct protective effect on the function and survivalof isolated human islets exposed to combined
proinflammatory and Th1 cytokines. J Clin Endocrinol
Metab 2001;86:4974-4978.
28. Martinez FO, Helming L, Gordon S: Alternative activation ofmacrophages: an immunologic functional perspective.
Annu Rev Immunol 2009;27:451-483.
29. Medzhitov R: Origin and physiological roles ofinflammation. Nature 2008;454:428–435.
30. Nguyen KD, Qiu Y, Cui X, et al: Alternatively activatedmacrophages produce catecholamines to sustain adaptive
thermogenesis. Nature 2011;480:104-108.
31. Odegaard JI, Ricardo-Gonzalez RR, Goforth MH, et al:
Macrophage-specific PPARgamma controls alternative
activation and improves insulin resistance. Nature
2007;447:1116-1120.
32. Oliver E, McGillicuddy F, Phillips C, et al:The role ofinflammation and macrophage accumulation in thedevelopment of obesity-induced type 2 diabetes mellitus andthe possible therapeutic effects of long-chain n-3 PUFA.
Proc Nutr Soc 2010;69:232–243.
33. Pepys M B, Hirschfield GM: C-reactive protein: a critical
update. J Clin Invest 2003;111(12): 1805-1812.
34. Rael EL, Lockey RF: Interleukin-13 Signaling and Its Rolein Asthma. World Allergy Org J 2011;4(3): 54-64.
35. Rosen ED, Spiegelman BM: Adipocytes as regulators ofenergy balance and glucose homeostasis. Nature 2006;
444:847–853.

36. Russell MA, Cooper AC, Dhayal S, et al: Differential effects
of interleukin-13 and interleukin-6 on Jak/STAT signaling andcell viability in pancreatic beta-cells. Islets 2013;5(2): 95-105.
37. Schmidt FM, Weschenfelder J, Sander C, et al: Inflammatory
cytokines in general and central obesity and modulating
effects of physical activity. PLoS One 2015;10(3):e0121971.
38. Schmitz J, Owyang A, Oldham E, et al: IL-33, an interleukin1-like cytokine that signals via the IL-1 receptor-related
protein ST2 and induces T helper type 2-associatedcytokines. Immunity 2005; 23(5):479-490.
39. Sell H, Habich C, Eckel J: Adaptive immunity in obesity andinsulin resistance. Nat Rev Endocrinol 2012;8(12):709-716.
40. Sica A, Mantovani A: Macrophage plasticity and polarization:in vivo veritas. J Clin Invest 2012;122(3):787-795.
41. Souza KL, Gurgul-Convey E, Elsner M, et al: Interaction
between pro-inflammatory and anti-inflammatory cytokines
in insulin-producing cells. J Endocrinol 2008;197:139-150.
42. Surendar J, Mohan V, Rao MM, et al: Increased levels ofboth Th1 and Th2 cytokines in subjects with metabolic
syndrome (CURES-103). Diabetes Technol Ther.
2011;13(4):477-482.
43. Trayhurn P: Endocrine and signalling role of adipose tissue:
new perspectives on fat. Acta Physiol Scand
2005;184:285-293.
44. Whittle AJ, Vidal-Puig A: Physiology: Immune cells fuel thefire. Nature 2011;480: 46-47.
45. Wu D, Molofsky AB, Liang HE, et al: Eosinophils sustain
adipose alternatively activated macrophages associatedwith glucose homeostasis. Science 2011;332: 243-247.
46. Xia S, Sha H, Yang L, et al: Gr-1+ CD11b+ myeloid-derived
suppressor cells suppress inflammation and promote insulin
sensitivity in obesity. J Biol Chem 2011;286(26):2359123599.

EVALUAREA NIVELULUI SERIC AL CITOKINEIANTI-INFLAMATOARE IL13 LA PACIENȚI OBEZIȘI CU DIABET ZAHARAT DE TIP 2
REZUMAT
Scop: Studiul nivelului circulant al IL13 la pacienții obezi și diabetici. Metode: În studiu au fost incluși 69 pacienți
împărțiți în patru subloturi: obezi non diabetici (O) (n= 24), obezi diabetici (OD) (n= 24), non obezi diabetici (NOD)(n= 9) și lot de control cu subiecți normoponderali fără diabet (C) (n= 12). Pentru toți pacienții incluși în studiu sau înregistrat date precum vârstă, sex, greutate, IMC, circumferință abdominală și a coapselor, tensiune arterială
și s-a determinat nivelul glucozei serice, hemoglobinei glicate, proteinelor totale și electroforeza proteinelor,parametrii profilului lipidic, ureea, creatinina, proteina C-reactivă și, IL13. Datele au fost analizate statistic pentrutoți pacienții incluși în studiu și separat pentru fiecare sublot. Rezultate: Nivelul IL13 a fost semnificativ crescut lapacienții obezi față de lotul de control (27.61±9.72 pg/ml vs 20.69±5.89 pg/ml,
p= 0.0327). S-a observat o corelație pozitivă moderată între nivelul IL13 și circumferința abdominală (coeficient decorelație rs 0.416, p=0.0004). Analiza de regresie multivariată a arătat că circumferința abdominală și nivelul IL4
au fost predictori semnificativi pentru nivelul IL13 (model de regresie p=0.0086). Nu s-au observat diferențe
statistice semnificative între nivelurile IL13 la subiecții din loturile NOD și C. Concluzii: Obezitatea abdominală afost asociată cu nivel crescut al IL13 care s-a corelat negativ cu cel al proteinei C reactive. La subiecții non obezi,
diabetul nu s-a asociat cu modificări ale nivelului seric al IL13.
Cuvinte cheie: nivelule seric al IL13, cytokine antiinflamatoare, obezitate, diabet

Fiziologia - Physiology • 2020.30.2(100)33

AN OVERVIEW ON THE USE OF PROBE-BASEDCONFOCAL LASER ENDOMICROSCOPY IN MODERNMEDICAL PRACTICE
ANDREEA-GEORGIANA GHEORGHE1*, FLORIN ANGHELINA2*, MARIUS
MATEI3, ALIN DRAGOȘ DEMETRIAN4, MIHAI OLTEANU5, COSTINTEODOR STREBA5,6
1Ph D Student, Department of Pulmonology
2Department of ENT, University of Medicine and Pharmacy of Craiova, Romania
3Department of Histology, University of Medicine and Pharmacy of Craiova, Romania
4Department of Thoracic Surgery, University of Medicine and Pharmacy of Craiova, Romania
5Department of Pulmonology, University of Medicine and Pharmacy of Craiova, Romania
6Department of Research Methodology, University of Medicine and Pharmacy of Craiova, Romania* = AG Gheorghe and F Anghelina contributed equally to this paper and thus share first authorship.

ABSTRACT:
Confocal laser endomicroscopy is a rather novel technique used to investigate the inner mucosa during endoscopic
or laparoscopic interventions. The principle was discovered several decades ago; however, only recent
technological advances allowed for a sufficient miniaturization of the components, necessary to transpose the idea
to reality. Probe-based confocal laser endomicroscopy was thus established as a way to obtain optical biopsies
during standard endoscopic exploratory procedures. We aimed to highlight the importance of the procedure in
obtaining a rapid, valid diagnosis, with sufficient accuracy, rapidity and cost savings to justify the necessary initialinvestment and additional expertise required for the task.
Keywords: confocal laser endomicroscopy, optical biopsy, digestive tract, respiratory system.

INTRODUCTION:
Confocal laser endoscopy is a new technique thatenable a histological examination (optical biopsy) to be
performed during any endoscopic procedure. This
helps to improve the diagnosis and define thetreatment needed for multiple digestive diseases [1].
The mode of operation of confocal endomicroscopy
was first described in 1957. A fiber optic probe
advances through the channel of a bronchoscope, isdirected to an area of interest that illuminates withlaser light (488 nm frequently used) and light reflected
is redirected in the opposite direction through an orifice[2-4].
The procedure is deemed as "confocal" since thelightning source and detection system are comprised
in one common focal plane [5], allowing for a focalized
view of one specific region from which classicalbiopsies can be taken [6]. This procedure allows for

rapid and precise identification of the areas of interest,
facilitating prior so-called “optical biopsies” of thetissue.
After fluorescein is administered, it can be performedin vivo even during the current endoscopic technique.
The use of contrasting agents – either fluorescein orother fluorescent dyes- facilitates imaging of cellular
structures, including nuclear and cytoplasmic
structures, thus revealing the mucosal patterns.
Barret’s esophagus or celiac disease andinflammatory diseases specific to the gastrointestinal
tract are one of the main applications that were initiallytackled through this procedure. Classical biopsies
were thus facilitated and pCLE showed that in vivo
imaging can be achieved at cellular level, hence
broadening the range of applications to neoplastic
diseases (especially malignant) of the entire digestive
tract – esophagus, stomach or colon – showing good
accuracy and sufficient specificity [7-15]

Received 5th of September 2020. Accepted 4th of October 2020. Address for correspondence: Marius Matei, MD PhD,
Department of Histology, University of Medicine and Pharmacy of Craiova, Romania; Craiova, Petru Rares Street no.2, Romania.
Phone: +40-723535854; e-mail: marius.matei@umfcv.ro34

Fiziologia - Physiology • 2020.30.2(100)AIM
The main objective of this short review is to show theimportance of using laser confocal endomicroscopy in
the identification of lesions in the digestive tract but
also the ability of this technique in detecting malignant
lung lesions, because it has the potential tofundamentally change the role of biopsy ingastrointestinal and especially lung endoscopy in thenear future.

OVERVIEW OF THE ADVANTAGES OF PCLE
Even though is considered a “gold standard”
diagnostic test, biopsy can be expensive in certain
settings and can generate sufficient delay between the
moment when endoscopy is performed and the time ofdiagnosis, as the transportation, preparation andinterpreting of the resulting tissue sample can be
prolonged by a number of factors. Even if theinterventional risk is lower than other procedures anda large number of biopsies are regularly performed
worldwide, it is not a procedure that can be safely
recommended in some cases, due to increased risk of
bleeding or the anatomical localization of the region[16]. Other restricting aspects can be related to theneed to perform it repeatedly, the incertitude of thetargeted area or the limited anatomical space.
Compared to these shortcomings of classicalbiopsies, pCLE offers various advantages:-decreases the risk of unwanted side effects byperforming a non-invasive microscopic analysis of thelesion;-functional evaluation of the structure (eg dynamic
alveoli, real-time elastin fiber, etc.);-quick diagnosis, by the presence of an
anatomopathologist in the bronchoscopy room;-possibility to enter more difficult areas.

THE IMPORTANCE OF PCLE IN DIGESTIVE
PATHOLOGY
It can be used in all parts of the gastrointestinal tractand is often helpful in identifying certain diseases suchas: Barret's esophagus, esophageal cancer,
pancreatic cystic neoplasms and biliary strictures,
ulcerative colitis, celiac disease, polyps, colorectal
cancer assessment, inflammatory bowel disease,detection in in vivo of Helicobacter pylori infection [17].
Comparing the results of the two procedures (pCLE
and histopathological examination) in the lesions of
the gastrointestinal tract, they demonstrated an
accuracy of about 86% to 96% [18].
The gold standard for the diagnosis of any diseaseremains the histopathological examination. However,the percentage of misdiagnosis can be significant due
Fiziologia - Physiology • 2020.30.2(100)to factors: misinterpretation, inaccuracy of the place ofthe biopsy or the number of biopsies performed.
The accuracy of this technique in gastric diseases was
94% to 96% for malignant diagnosis, compared tohistological biopsies and 88% for premalignant
diseases (such as intestinal metaplasia) [19].
In the colon, CLE had an accuracy of 82% when trying
to predict the type of histology in the case of polyps
when examining in-vivo; when used in conjunction withdigital chromoendoscopy with narrowband imaging
during surgery, its accuracy increased to 94% [20].
In inflammatory bowel disease (IBD), several studies
have examined the role of CLE in patient surveillance,
analyzing the degree of disease, biopsies, early
detection of dysplasia, evaluation of mucosal healing
and helping to define treatment stages [21, 22].
New applications have also been studied in the biliary
tract for the diagnosis of different subtypes ofpancreatic cysts, which have an average accuracy ofapproximately 85% for neoplastic and non-neoplastic
lesions [23, 24]

THE USE OF PCLE IN RESPIRATORYPATHOLOGY
Lung cancer is undeniably the most common
malignancy in the world. Globally, lung cancer casesand deaths are increasing. In 2018, GLOBOCAN
estimated 2.09 million new cases and 1.76 million
deaths, becoming the most common cause of cancer
deaths in men and women combined. It is a disease
whose diagnosis can be difficult and often at the timeof diagnosis is already in a metastatic stage. If it isdetected at an early stage, before it is spread, there isa greater chance that it will be treated successfully.
Although screening technology is constantly
advancing, research still shows that the death ratefrom lung cancer has not improved. Most lung cancers
are diagnosed in an advanced stage [25]. Its detection
is not nowadays a routine practice. The disease cansometimes be detected in the early stages by various
tests and analyzes that are performed for completely
different reasons.
The most common methods used to detect lungcancer include: lung radiography, chest CT scan,bronchoscopy, sputum cytology.
The diagnosis of lung cancer must include histologicalconfirmation, the degree of tumor spread (staging) but
also the analysis of the patient's functional status todecide treatment options [26].
Probe-based laser confocal endomicroscopy is a fairly
new technique respiratory medicine. It is an imaging
technique that can be used during bronchoscopy andthoracoscopy [27, 28]. The airways, pleura, lungtumors and lymph nodes can be viewed in real time35

with a resolution of up to 3.5 µm and a maximum depthof 70 µm, with a field of view of up to 600 nm maximum.
Through flexible bronchoscopy, it is possible tovisualize in real time, in vivo, the elastin microstructure
existing in the walls of the airways, but also of thealveoli. The procedure is performed under moderate
sedation. This technique has the advantage ofproviding high quality images, without major risks, andwithout significant morbidity. It also allows noninvasive
diagnosis. Also, with the help of this technique it ispossible to monitor the response to treatment andpossibly even the progression of the disease.
However, pCLE has not yet reached its full potential
and development is still needed. Although confocal
endomicroscopy is about to enter the usual practice ingastroenterology, in pulmonary medicine, it is still
limited, there are only studies on small groups ofpatients [29].
Various studies have proved that pCLE can identify
abnormal areas in endobronchial [29-34] andparenchymal malignancies (for both primary andmetastatic neoplasm) [35-37]. Filner et al. [34]demonstrated the ability of pCLE to differentiate
between normal lesions compared to neoplastic
lesions and vice versa in endobronchial lesions.
In their study [38], performed on 32 patients suspectedof lung cancer, Fuchs et al compared data from 75,522
images accumulated by pCLE with pathological dataobtained from biopsy samples. In this way, they wereable to identify different patterns, specific toneoplasms: chaotic and non-overlapping distributionof cells, changes in nucleus - heterogeneous size andfluctuating distances between them. Acriflavin, on theother hand, was not homogeneously absorbed by theneoplastic tissue, leaving areas without absorption.
According to these data, the study supports highaccuracy for pCLE (91%), neoplastic changes being
detected with a sensitivity of 96% and a specificity of
87.1% - because it allowed the differentiation ofcellular and subcellular elements and underground
imaging. Thus, a clear differentiation was obtained
between inflammatory infiltrates, normal mucosa andmalignant elements.
Additional and inspiring attempts were made by
applying CLE to separate normal mucosa fromcancerous tissue, both in vivo and ex vivo [39, 40], butalso alveolar lung microlithiasis [41] and radiationinduced pulmonary fibrosis [42]. CLE has been shown
to be useful for accurately highlighting the peripherallung nodule [43], but also the solitary lung nodules
highlighted in all segments of both lungs [44]. Guisieret al. demonstrated the successful application of CLE
for apoptosis to tumor xenografts in mice [45].
In her study, Anastasia Sorokina et al [46] presented
the collection of 18 lobectomy specimens from 18
different patients. Images of lung cancer samples and36

microscopy samples prepared from the same pCLE
samples were compared. It has been shown that pCLE
can detect lung carcinoma in tissues ex vivo fromneoplasia areas of lung tissue samples
Sabine Zirlik et al [47] analyzed CLE as a new
diagnostic route for differentiating malignant and nonmalignant pleural effusions. Thoracentesis was
performed in 100 patients with pleural effusions.
Cresyl violet and acriflavine were used as contrast
agents. CLE was able to identify malignant cells in
pleural effusions. Sensitivity was 87% (p: 87%) and 81%(p: 72%) for acriflavin and cresyl violet respectively. In
terms of specificity, they obtained an average value of
99% and 92%.
In a study of 91 patients identified with a peripherallung nodule [48], it was found that although in a fairly
high percentage (92%) the pCLE could identify areasof abnormal tissue, it could not discern malign frombenign areas. Several authors believe that thisoccurred because fluorescein does not penetrate sowell into the epithelial layer of the bronchi [49,50] andthus it is difficult to stain epithelial cells in the bronchial
mucosa [48, 51-54].
Bonhomme et al [55] presented three cases withpulmonary pleural pathology using pCLE (three
images of the pleura were presented: normal pleura,
non-cellular lung cancer with pleural metastasis and amesothelioma). pCLE showed a clear differentiation of
normal from malignant pleura in these cases.

SAFETY AND COSTS
When taking into account the necessary procedural
steps, complications during CLE can happen either
when the intravenous fluorescein is administered
before performing CLE and during the evaluation ofmini-samples from the selected tissue. CLE requires
the use of a fluorophore, a dye that is fluorescent whenstimulated by the laser beam, allowing the contrast to
intensify [56]. However, initial in vivo studies that
followed pCLE concluded that it was safe and deemed
it to be well tolerated by the awake patient, with local
anesthesia only and without the requirement of
supplemental oxygen administration. In addition,
contrary to the significant bleeding episodes that
accompanied acinar imaging, pCLE did not causesignificant discomfort or adverse effects [57].
pCLE has been proven to have higher performanceelements in assessing injuries, possibly reducing theneed for repeated procedures and lowering costs.

FUTURE PERSPECTIVES
New studies focusing on resolving relative barriers to
the use of CLE are currently needed. The results thathave been obtained in recent years demonstrate the
Fiziologia - Physiology • 2020.30.2(100)use of CLE in clinical practice, and a first step in thisregard could be the treatment of patients withuncertain diagnoses. In addition to improving andresolving many unclear diagnoses, therapeutic
decisions and / or follow-up procedures in this type of
patient may also be improved [58].

CONCLUSION
Confocal laser endomicroscopy is a procedure thathas demonstrated its ability to accurately diagnose
and therefore may be helpful in daily practice toincrease the ability to diagnose and manage thetreatment of patients.
The somewhat prohibitive initial costs to implement thesystem in some settings still prevent the widespreaduse of the procedure, along with the fact that it is not
implemented in normal countrywide strategies of manystates; however, recent regulatory advancements
gave way to its introduction into some developed
countries. Total or partial reimbursement and its clear
indication in several guidelines, along with effective
training programs for both performing the procedureand for image interpretation, further pushed theprocedure worldwide.
All limitations are thus relative, with easy to implement
solutions that can show its use can largely overcome
the initial difficulties and thus warrant its widespread
implementation.
Acknowledgements
This work was supported by a grant of the Romanian
Ministry of Education and Research, CNCS UEFISCDI, project number PN-III-P1-1.1-TE-20191474, within PNCDI III
Conflicts of interest
The authors declare no conflicts of interests

REFERENCES
1.

2.

3.

Tahara T, Horiguchi N, Terada T, Yamada H, Yoshida
D, Okubo M, Funasaka K, Nagasaka M, Nakagawa Y,
Tsukamoto T, Shibata T, Kuroda M, Ohmiya N.
Diagnostic utility of probe-based confocal laser
endomicroscopy in superficial non-ampullary
duodenal epithelial tumors. Endosc Int Open. 2019;7(11):E1515-E1521.
Thiberville L, Salaün M, Bourg-Heckly G. In vivo
confocal microendoscopy: from the proximal bronvhus
down to the pulmonary acinus. Eur Respir Mon. 2010;
48: 73-79.
Thiberville L, Salaün M. Diagnostic of lung cancer:
confocal bronchoscopy. In: Diaz-Jimenez JP,
Rodriguez AN, editors. Interventions in pulmonary
medicine. Springer New York. 2013: 221-230.

Fiziologia - Physiology • 2020.30.2(100)4.

Vercauteren T. Image Registration and Mosaicing for
Dynamic In Vivo Fibered Confocal Microscopy. PhD
Thesis, Paris. 2007
5. High-definition
andhigh-magnification
endoscopes.Gastrointestinal Endoscopy. 2014. 80(6) :919-927.
6. Levent Dalar, Cengiz Özdemir, Sinem Nedime Sökücü,
Ahmet Levent Karasulu, Sedat Altin, Neslihan Fenerand Filiz Koşar. Probe-based Confocal Laser
Endomicroscopy (pCLE) in the Diagnosis of Diffuse
Parenchymal Lung Diseases: Two Cases. Austin J
Pulm Respir Med. 2014. 1(4): 1018
7. Fuchs FS, Zirlik S, Hildner K, Schubert J, Vieth M,
Neurath MF. Confocal laser endomicroscopy for
diagnosing lung cancer in vivo. Eur Respir J.
2013;41(6):1401-8.
8. Polglase AL, McLaren WJ, Skinner SA, etal, Afluorescence confocal endomicroscope for invivo microscopy of the upper- and the lower-GI
tract. Gastrointest Endosc 2005; 62: 686–695.
9. Kiesslich R, Burg J, Vieth M, et al. Confocal laser
endoscopy for diagnosing intraepithelial neoplasias
andcolorectal
cancer invivo. Gastroenterology 2004; 127: 706–713.
10. Kakeji Y, Yamaguchi S, Yoshida D, et
al. Development and assessment of morphologic
criteria for diagnosing gastric cancer using confocal
endomicroscopy:
an exvivo and invivo study. Endoscopy 2006; 38: 886–890.
11. Neumann H, Kiesslich R, Wallace MB, et al. Confocal
laser endomicroscopy: technical advances and clinicalapplications. Gastroenterology 2010; 139: 388–392.
12. Bojarski C, Gunther U, Rieger K, etal,In
vivo diagnosis of acute intestinal graft-versus-hostdiseasebyconfocalendomicroscopy. Endoscopy 2009; 41: 433–438.
13. Haxel BR, Goetz M,Kiesslich R, et
al. Confocal
endomicroscopy: a novel application for imaging oforal and oropharyngeal mucosa in human. Eur Arch
Otorhinolaryngol 2010; 267: 443–448
14. Sanduleanu S, Driessen A, Gomez-Garcia E, et al. In
vivo diagnosis and classification of colorectal
neoplasia by chromoendoscopy-guided confocal laser
endomicroscopy. Clin
Gastroenterol
Hepatol 2010; 8: 371–378
15. Venkatesh K, Cohen M, Evans C, et al. Feasibility ofconfocal endomicroscopy in the diagnosis of pediatricgastrointestinal
disorders. WorldJ
Gastroenterol 2009; 15: 2214–2219
16. Wallace M et al. Miami classification for pCLE.
Endoscopy. 2011. 43(10):882-91
17. Kiesslich R, Goetz M, Neurath MF. Confocal laser
endomicroscopy for gastrointestinal diseases.
Gastrointest Endosc Clin N Am. 2008. 18(3):451-66.

37

18. Robles-Medranda C. Confocal endomicroscopy: Is it
time to move on?. World J Gastrointest Endosc.
2016;8(1):1-3.
19. Lim LG, Yeoh KG, Srivastava S, Chan YH, Teh M, Ho
KY. Comparison of probe-based confocal
endomicroscopy with virtual chromoendoscopy andwhite-light endoscopy for diagnosis of gastric intestinal
metaplasia. Surg Endosc. 2013;27:4649–4655.
20. Shahid MW, Buchner AM, Heckman MG, Krishna M,
Raimondo M, Woodward T, Wallace MB. Diagnostic
accuracyofprobe-based
confocallaser
endomicroscopy and narrow band imaging for small
colorectal polyps: a feasibility study. Am J
Gastroenterol. 2012;107:231–239.
21. Neumann H, Vieth M, Atreya R, Neurath MF, Mudter J.
Prospective evaluation of the learning curve ofconfocal laser endomicroscopy in patients with
IBD. Histol Histopathol. 2011;26:867–872.
22. Kiesslich R, Goetz M, Lammersdorf K, Schneider C,
Burg J, Stolte M, Vieth M, Nafe B, Galle PR, Neurath
MF. Chromoscopy-guided endomicroscopy increases
the diagnostic yield of intraepithelial neoplasia inulcerative colitis. Gastroenterology. 2007;132:874–
882.
23. Slivka A, Gan I, Jamidar P, Costamagna G, Cesaro P,
Giovannini M, Caillol F, Kahaleh M. Validation of thediagnostic accuracy of probe-based confocal laser
endomicroscopy for the characterization ofindeterminate biliary strictures: results of a prospective
multicenterinternational
study. Gastrointest
Endosc. 2015;81:282–290.
24. Napoléon B, Lemaistre AI, Pujol B, Caillol F,
Lucidarme D, Bourdariat R, Morellon-Mialhe B, Fumex
F, Lefort C, Lepilliez V, Palazzo L, Monges G, Filoche
B, Giovannini M. A novel approach to the diagnosis ofpancreatic serous cystadenoma: needle-based
confocallaser
endomicroscopy. Endoscopy. 2015;47:26–32.
25. Ganti AK, Mulshine JL. Lung Cancer Screening. The
Oncologist .2006. 11(5):481-7.
26. Stefan Hammerschmidt, Hubert Wirtz. “Lung Cancer:
Current Diagnosis and Treatment. Dtsch Arztebl Int.
2009. 106(49): 809–820.
27. Wijmans L, Baas P, Sieburgh TE, de Bruin DM, Ghuijs
PM, van de Vijver MJ, et al. Confocal Laser
Endomicroscopy as a Guidance Tool for Pleural
Biopsies in Malignant Pleural Mesothelioma. Chest.
2019, Epub ahead of print.
28. Goorsenberg A, Kalverda K, A, Annema J, Bonta P:
Advances in Optical Coherence Tomography andConfocal Laser Endomicroscopy in Pulmonary
Diseases. Respiration 2020;99:190-205.
29. Yserbyt J, Dooms C, Ninane V, Decramer M, Verleden
G. Perspectives using probe-based confocal laser
endomicroscopy of the respiratory tract. Swiss Med
Wkly. 2013
38

30. Thiberville L, Moreno-Swirc S, Vercauteren T, Peltier
E, Cavé C, Bourg Heckly G. In vivo imaging of thebronchial wall microstructure using fibered confocal
fluorescence microscopy. Am J Respir Crit Care Med.
2007 Jan;175(1):22–31.
31. Wellikoff AS, Holladay RC, Downie GH, Chaudoir CS,
Brandi L, Turbat-Herrera EA. Comparison of in vivo
probe-based confocal laser endomicroscopy withhistopathology in lung cancer: A move toward opticalbiopsy. Respirology. 2015 Aug;20(6):967–74.
32. Shah PL, Kemp SV, Newton RC, Elson DS, Nicholson
AG, Yang GZ. Clinical Correlation between Real-Time
Endocytoscopy, Confocal Endomicroscopy, andHistopathology in the Central Airways. Respiration.
2017;93(1):51–7.
33. Fuchs FS, Zirlik S, Hildner K, Schubert J, Vieth M,
Neurath MF. Confocal laser endomicroscopy for
diagnosing lung cancer in vivo. Eur Respir J. 2013
Jun;41(6):1401–8.
34. Filner JJ, Bonura EJ, Lau ST, Abounasr KK, Naidich D,
Morice RC, et al. Bronchoscopic fibered confocal
fluorescence microscopy image characteristics andpathologic correlations. J Bronchology Interv
Pulmonol. 2011 Jan;18(1):23–30.
35. Hassan T, Piton N, Lachkar S, Salaün M, Thiberville L.
A Novel Method for In Vivo Imaging of Solitary Lung
Nodules Using Navigational Bronchoscopy andConfocal Laser Microendoscopy. Lung. 2015
Oct;193(5):773–8.
36. Sorokina A, Danilevskaya O, Averyanov A, Zabozlaev
F, Sazonov D, Yarmus L, et al. Comparative study of
ex vivo probe-based confocal laser endomicroscopy
and light microscopy in lung cancer diagnostics.
Respirology. 2014 Aug;19(6):907–13.
37. Goorsenberg A, Kalverda K, A, Annema J, Bonta P:
Advances in Optical Coherence Tomography andConfocal Laser Endomicroscopy in Pulmonary
Diseases. Respiration 2020;99:190-205.
38. Florian S Fuchs, Sabine Zirlik,Kai Hildner, Juergen
Schubert, Confocal laser endomicroscopy for
diagnosing lung cancer in vivo, European Respiratory
Journal 41(6)39. Fuchs FS, Zirlik S, Hildner K, et al. Confocal laser
endomicroscopy for diagnosing lung cancer invivo. Eur
Respir
J. 2013;41(6):1401–
8. [PubMed] [Google Scholar]
40. Sorokina A, Danilevskaya O, Averyanov A, et al.
Comparative study of ex vivo probe-based confocal
laser endomicroscopy and light microscopy in lungcancer diagnostics. Respirology. 2014;19(6):907–13
41. Yserbyt J, Alame T, Dooms C, Ninane V. Pulmonary
alveolar microlithiasis and probe-based confocal laser
endomicroscopy. J
Bronchology
Interv
Pulmonol. 2013;20(2):159–63.
42. Perez JR, Ybarra N, Chagnon F, et al. Image-guided
fluorescence endomicroscopy: From macro- to microFiziologia - Physiology • 2020.30.2(100)imaging of radiation-induced pulmonary fibrosis. Sci
Rep. 2017;7(1):17829.
43. Su Z, Zhong C, Li S, et al. Needle-based confocal laser
endomicroscopy in the diagnosis of peripheral
pulmonary nodule: A preliminary report. J Thorac
Dis. 2017;9(8):2608–12.
44. Hassan T, Thiberville L, Hermant C, et al. Assessing
the feasibility of confocal laser endomicroscopy insolitary pulmonary nodules for different part of thelungs, using either 0.6 or 1.4 mm probes. PLoS
One. 2017;12(12):e0189846.
45. Guisier F, Bohn P, Patout M, et al. In- and exvivo molecular imaging of apoptosis to assess
sensitivity of non-small cell lung cancer to EGFR
inhibitors using probe-based confocal laser
endomicroscopy. PLoS One. 2017;12(7):e0180576.
46. Anastasia Sorokina, Olesya Danilevskaya, Alexander
Averyanov, Fedor Zabozlaev, Dmitry Sazonov, Lonny
Yarmus4 And Hans J. Lee4 . Comparative Study Of Ex
Vivo Probe-Based Confocal Laser Endomicroscopy
And Light Microscopy In Lung Cancer Diagnostics.
Respirology. 2014. 19: 907–913.
47. Zirlik S, Hildner K, Rieker RJ, Vieth M, Neurath MF,
Fuchs FS. Confocal Laser Endomicroscopy for
Diagnosing Malignant Pleural Effusions. Med Sci
Monit. 2018;24:5437-5447. Published 2018 Aug 5.
48. Seth S, Akram AR, McCool P, Westerfeld J, Wilson D,
McLaughlin S, Dhaliwal K, Williams CK. Assessing theutility of autofluorescence-based pulmonary opticalendomicroscopy to predict the malignant potential ofsolitary pulmonary nodules in humans. Sci Rep. 2016
Aug 23;6:31372.
49. Fuchs FS, Zirlik S, Hildner K, Frieser M, Ganslmayer
M, Schwarz S et al. Fluoresceinaided confocal laser
endomicroscopy of the lung. Respiration.
2011;81(1):32–8.
50. Newton RC, Kemp SV, Yang GZ, Elson DS, Darzi A,
Shah PL. Imaging parenchymal lung diseases with

Fiziologia - Physiology • 2020.30.2(100)confocal endomicroscopy. Respir Med. 2012
Jan;106(1):127–37.
51. Thiberville L, Moreno-Swirc S, Vercauteren T, Peltier
E, Cavé C, Bourg Heckly G. In vivo imaging of thebronchial wall microstructure using fibered confocal
fluorescence microscopy. Am J Respir Crit Care Med.
2007 Jan; 175(1):22–31.
52. Shah PL, Kemp SV, Newton RC, Elson DS, Nicholson
AG, Yang GZ. Clinical Correlation between Real-Time
Endocytoscopy, Confocal Endomicroscopy, andHistopathology in the Central Airways. Respiration.
2017;93(1):51– 7
53. Sorokina A, Danilevskaya O, Averyanov A, Zabozlaev
F, Sazonov D, Yarmus L, et al. Comparative study of
ex vivo probe-based confocal laser endomicroscopy
and light microscopy in lung cancer diagnostics.
Respirology. 2014 Aug;19(6):907–13.
54. Goorsenberg A, Kalverda KA, Annema J, Bonta P.
Advances in Optical Coherence Tomography andConfocal Laser Endomicroscopy in Pulmonary
Diseases. Respiration. 2020;99(3):190-205.
55. Bonhomme O, Duysinx B, Heinen V, Detrembleur N,
Corhay JL, Louis R. First report of probe based
confocal laser endomicroscopy during medical
thoracoscopy. Respir Med. 2019 Feb;147:72–5
56. Kwan AS, Barry C, McAllister IL, Constable I.
Fluorescein angiography and adverse drug reactions
revisited: the Lions Eye experience. Clin Experiment
Ophthalmol. 2006;34:33–38.
57. Levent Dalar, Cengiz Özdemir, Sinem Nedime Sökücü,
Ahmet Levent Karasulu, Sedat Altin, Neslihan
Fener and Filiz Kosar, Probe-based Confocal Laser
Endomicroscopy (pCLE) in the Diagnosis of Diffuse
Parenchymal Lung Diseases: Two Cases, Austin J
Pulm Respir Med 2014;1(4): 1018.
58. Carlos Robles-Medranda, Confocal endomicroscopy:
Is it time to move on? World J Gastrointest Endosc.
2016 Jan 10; 8(1): 1–3.

39

O PREZENTARE GENERALĂ A UTILIZĂRIIENDOMICROSCOPIEI CONFOCALE LASER BAZATEPE SONDĂ ÎN PRACTICA MEDICALĂ MODERNĂREZUMAT
Endomicroscopia confocală laser este o tehnică relativ nouă utilizată pentru a investiga mucoasa în timpul
intervențiilor endoscopice sau laparoscopice. Principiul a fost descoperit în urmă cu câteva decenii; cu toate
acestea, doar progresele tehnologice recente au permis o miniaturizare suficientă a componentelor, necesaretranspunerii ideii în realitate. Endomicroscopia confocală laser pe bază de miniprobe a fost astfel stabilită ca omodalitate de a obține biopsii optice în timpul procedurilor exploratorii endoscopice standard. Am urmărit să
evidențiem importanța procedurii în obținerea unui diagnostic rapid și valid, cu precizie suficientă, rapiditate și
economii de costuri pentru a justifica investiția inițială și experiența suplimentară necesară.
Cuvinte cheie: endomicroscopia confocală laser, biopsie optică, tract digestiv, sistem respirator.

40

Fiziologia - Physiology • 2020.30.2(100)

CLINICALAPPLICATIONSOFBASOPHIL
ACTIVATION TEST IN ALLERGY DIAGNOSISMARIA-ROXANA BUZAN1,2, DANIELA CRÎSNIC1,2,3, MANUELA GRIJINCU1,2, LAURIANA-EUNICEZBÎRCEA1,2, TUDOR PAUL TAMAȘ1,2, MONICA DANIELA COTARCĂ1,2, LAURA HAIDAR1,2, GABRIELATĂNASIE1,2, CARMEN PANAITESCU1,2, KUAN-WEI CHEN2
1Department of Functional Sciences, „Victor Babeș” University of Medicine and Pharmacy Timișoara, România
2OncoGen - Centre for Gene and Cellular Therapies in the Treatment of Cancer, „Pius Brînzeu” Clinical Emergency County HospitalTimișoara, România
3SC Biodim SRL, Ciprian Porumbescu Street, Timișoara, România

ABSTRACT
Currently, IgE-mediated allergy diagnosis is based mainly on skin prick tests, but these tests are not recommended
for patients that had previously experienced a severe allergic reaction to an allergen source. Serum specific IgE
quantification has also its flaws. Therefore, in vitro cellular assays, such as the basophil activation test (BAT) area good diagnosis alternative. Basophil activation test measures the IgE-mediated hypersensitivity to allergens
without exposing the patients to a risk of anaphylaxis. BAT is a functional assay that measures the expression ofactivation markers such as CD63 and CD203c on the surface of basophils. It is widely used in allergy research but
recent clinical validations and standardisations made this test available also for routine testing. In this study, we
describe the mechanisms behind the basophil activation test and its clinical application in food, respiratory, venom
and drug allergy diagnosis.
Keywords: Allergy, Basophil activation test, Allergy diagnosis, CD63, CD203c

INTRODUCTION
IgE-mediated hypersensitivity reactions are usuallydiagnosed after a careful clinical anamnesis andconfirmed by skin prick testing, measurements ofserum specific IgE and, when necessary, provocation
tests is. Skin testing performed with commerciallyavailable extracts is not always possible (e.g. antihistamines intake) or can be contraindicated (historyof life-threatening anaphylaxis). Furthermore,challenge tests cannot be recommended in daily
practice due to ethical considerations, practicalreasons and variations in sensitivity and specificity [1].
Therefore, clinicians confirm their clinical suspicions
after specific IgE quantification. Unfortunately, theseassays do not have absolute diagnostic reliability,
especially when it comes to pan-allergens with highpotential of cross-reactivity. Also, there might be clear
discrepancies between sensitization, revealed byserum IgE and the clinical manifestation of thatsensitization [1]. Basophil activation test is a good in

vitro alternative, mimicking an allergic reaction in a test
tube. It determines the ability of an allergen to induce
basophil activation, measuring the expression of
specific markers using flow cytometry. BAT has theadvantage of being a functional test, positive results
occur only after successful IgE cross-linking, not bymonovalent binding as in IgE assays [2]. In this review,
we describe the principle of basophil activation test
and its clinical applications in diagnosing food,respiratory, venom and drug allergy.

Received 12th of September 2020. Accepted 11th of October 2020. Address for correspondence: Buzan Maria-Roxana, OncoGenCentre for Gene and Cellular Therapies in the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency County Hospital Timisoara,
Romania, Liviu Rebreanu No.156 Blv., Timisoara, RO-300723, Romania; phone/fax: +40356433282; e-mail: buzan.roxana@umft.roFiziologia - Physiology • 2020.30.2(100)41

activation is a process that starts with intracellularchanges (signal transduction from FcεRI by means ofphosphorylation of p38MAPK and calcium influx,
followed by changes on the cell membrane level(CD203c) with final degranulation of the basophils(CD63) [1](Figure 2).

Fig. 1. Diagnostic algorithm for allergic disease(adapted after Hoffmann et al, 2015 [3])

BASOPHIL ACTIVATION TEST PRINCIPLES
The main characteristic of allergic eﬀector cells in
immediate-type allergy is allergen-speciﬁc IgE boundto the high aﬃnity IgE receptor, i.e., FcεRI, from thecell surface. Binding of allergens to surface IgE resultsin FcεRI cross-linking and elicits the acute phase of theallergic response that causes the sudden release ofvasoactive mediators into the tissue and/or circulation.
This process is called anaphylactic degranulation (andmay induce life-threatening anaphylaxis)[3]. In
contrast, in piecemeal degranulation, cells secrete
granule contents without exocytosis [4]. Both mast
cells and basophils share these key characteristics.
Mast cells reside in the tissue and are consideredprimary allergic eﬀector cells [5]. Basophils are
peripheral blood granulocytes, with secretory granules
containing histamine and are capable of secreting
proteases, cytokines, chemokines, and lipid
mediators. Basophils present on their surface uniquemarkers depending upon whether the cells undergo
anaphylactic or piecemeal degranulation, which canthen be measured by flow cytometry [6]. Basophil

42

Fig. 2. Comparison between a resting and astimulated basophil. Cross-linking of membranebound IgE from basophil induces mediators secretionand up-regulates the expression of specific activation
markers such as CD63 and CD203c (modified afterHausmann et al, 2009 [7]).
Basophil activation test measures an in vitro activationof patients’ basophils in the test tube. After blood
sample preparations (whole-blood assay or leukocyte
isolation) and preincubation with interleukin IL-3
(depending on the allergen of interest) or prewarmingof blood samples and reagents to 370C, the allergen is
added, and basophils are incubated for 15 minutes to
1 hour at 370C in the water bath (depending on theallergen of interest) (Stimulation). Crude allergen
extract, purified or recombinant allergens can be usedas stimulants. After stimulation, the samples arestained with monoclonal antibodies coupled tofluorochromes (Staining). Another step is the lysis ofcontaminating erythrocytes (Red blood cell lysis),
followed by data acquisition (using a flow cytometer)and data analysis (dedicated software) (Figure 3).

Fiziologia - Physiology • 2020.30.2(100)Fig. 3. Basophil activation test includes several steps: stimulation, staining, red blood cell lysis, data acquisition andinterpretation

The minimal number of basophils to be analyzed
should be 200 per test tube for accurate quantificationof basophil activation, but in order to minimize thevariation of results caused by equipment
measurement error, it is preferred that 600 to 1000
basophils to be evaluated. To select basophils fromthe whole leukocyte population, several positive andnegative selection strategies are possible. CD123,
CCR3, CRTH2, IgE can be used as identification
markers, while CD63, CD203c, CD107a and b, CD13,
CD164, CD69, p38 MAPK, STAT5 represent
activation markers (Table I) [7].

Table I. Basophil activation test identification and activation markers (modified after Hemmings et al, 2018 [2])Identification
markers

Activation
markers

MarkerCell expression

Description

CCR3

Basophils, mast cells,
Th2 lymphocytes

Stable marker for identification[8], [9]CD123

Basophils,
plasmacytoid dendritic
cells
Basophils, monocytes,
dendritic cells

Highly expressed in these cells
[10]

IgE
CRTH2

Basophils, eosinophils,
Th2 lymphocytes

CD63

Basophils, mast cells,
platelets, macrophages

Fiziologia - Physiology • 2020.30.2(100)Other cells expressing highaffinity IgE receptor FcɛRI [9],[11]Differentiation from eosinophils
by side scatter or T lymphocytes
by CD3 is reported [12]Widely used activation marker
and an accurate marker ofanaphylactic degranulation [13]43CD203c

Basophils, mast cells,
CD34+ progenitor cells

CD107a,CD107bBasophils, mast cells, T
cells and NK cells

CD13

Basophils, myeloid
cells

CD164

Basophils,
CD34+ progenitor cells

CD69

Basophils,
lymphocytes,
neutrophils,
monocytes, eosinophils
Various cell types

p38
MAPK,
STAT5

Currently, BAT diagnostic kits use CD63 asactivation marker (Flow CAST®, Buhlmann
Diagnostics Corp), while some kits measure
CD203c (Allergenicity Kit, Beckman Coulter).
CD63 is a 53kDa protein from the transmembrane4 superfamily, known also as lysosomal-associatedmembrane glycoprotein-3 (LAMP-3). When it comesto a resting basophil, this protein is located on themembrane of intracellular secretory granules. After
stimulation by FcɛRI, these granules fuse with theplasma membrane and thus, CD63 is expressed on
the surface of degranulated basophils [13]. Early
studies suggested that this marker is associatedwith granules containing histamine, suggesting that
CD63 up-regulation can be used as an indirect
marker of histamine release [13][18]. However,recent studies suggest that histamine release may
be the sum of both anaphylactic and piecemeal
degranulation, and up-regulation of CD63 may be
representative of only anaphylactic degranulation[19].
CD203c is another basophil activation marker
present on CD34+ progenitor cells, basophils andmast cells. It is a glycosylated type IItransmembrane molecule that belongs to the familyofectonucleotide
pyrophosphatase/phosphodiesterase
enzymes[20]. CD203c is constitutively expressed in low
levels on the basophil surface membrane and is44

Upregulation has been reported
to be representative ofpiecemeal degranulation also
used as identification marker
and expressed in low levels of
resting cells [14]CD107a and CD107b was foundto be only expressed by
activated basophils and its
upregulation was similar to
CD63.
CD13 and CD164 has an
expression profile comparable toCD203c [15]Expressed progressively whenstimulated with IL-3; however,found to be weakly expressed to
IgE mediated stimulation [16]Basophilic phosphorylation of
intracellular molecules canalternatively be used to measurebasophil activation [17]quickly upregulated upon cell activation via allergen(Figure 2) or more slowly via IL-3 [21]. This upregulation differs from CD63 in terms of the inciting
stimuli and early signalling events, which suggeststhat CD203c may be associated with piecemeal
degranulation [6].
The quality of basophils obtained is confirmed using
for stimulation fMLP, a bacterial peptide. Anti-IgE
antibodies can be used as IgE-mediated positive
control, while buffer can be used as a negative
control. The results of BAT can be displayed as apercentage of basophils expressing the defined
activation marker (in the case of CD63) or in termsof mean fluorescence intensity (MFI) (in the case ofCD203c), by calculating the stimulation index (SI),represented by the ratio between MFI of the sample
and the MFI of the negative control [22].

BAT APPLICATION IN FOOD ALLERGYWhen it comes to diagnosing food allergy, oral foodchallenge (OFC) is the gold standard. However, it
requires ingestion of food that can induce an acuteallergic reaction and needs to be performed in asupervised environment, capable of treating
anaphylaxis. Thus, specialists prefer, when it ispossible, to diagnose food allergy based on clinicalhistory, skin prick test and/or serum specific IgE.
Fiziologia - Physiology • 2020.30.2(100)These tests have high sensitivity but low specificity.
BAT can improve the diagnosis of food allergy proving
to be more accurate than IgE sensitization tests with a
specificity ranging between 75 and 100% andsensitivity between 77 and 98% [23]. This highspecificity may be able to reduce the number of OFC
performed. Also, BAT can distinguish between
individuals that are clinically allergic to those who aretolerant.
Basophil activation test is also used for monitoring theclinical response to immune-modulatory treatment offood allergy. Studies with immunotherapy for peanut
and egg show that the basophil reactivity to thoseallergens decreased during the treatment [24, 25].

BAT APPLICATION
ALLERGYIN

RESPIRATORY

Skin prick tests and serum levels of specific IgE,
combined with clinical history, were consideredsufficient to diagnose respiratory allergies, but allergic
reactions to inhaled allergens can be complex due tothe diversity of potential sources to which the patientsare exposed to and the number of tissues that can beaffected. Therefore, patients who report local allergic
rhinitis can have negative skin prick tests andundetectable levels of specific IgE, being hard todifferentiate between allergic and non-allergic rhinitis.
Despite the absence of serum allergen-specific IgE,
for these patients, BAT has proved more sensitive andable to diagnose IgE-mediated allergy [26].
Since basophil sensitivity is a reproducible measure[27] it can be used to determine the efficacy ofallergen-specificimmunotherapy(AIT)toaeroallergens. BAT was used for monitorisation ofpatients undergoing AIT with birch [28] and timothy[29] and showed reduced allergen sensitivity during
the treatment. This reduction may be due to blocking
factors competing (specific IgG) for the allergen withthe cell-bounded specific IgE [28], [29].

BAT APPLICATION IN VENOM ALLERGY
In the case of Hymenoptera venom allergy diagnosis,
basophil activation test report both high specificity (83100%) and sensitivity (85-100%) [30]–[32]. And eventhough testing for specific IgE to the major bee andwasp allergens (Api m 1 and Ves v 5) solves manysuspect cases, BAT is used in diagnosing uniqueissues. For example, a small percentage of patientswith a clinical history of venom allergy report
undetectable specific IgE and negative skin tests.
Since sting provocation tests are complicated andFiziologia - Physiology • 2020.30.2(100)have an unethical nature, BAT has proven effective for
80% of these patients [33].
Another issue with the diagnosis of Hymenoptera
venom allergy is the double-positive results for bee
and wasp venom, approximately 60% of patients
exhibit specific IgE to both. This double positivity may
be caused by a co-sensitization to both venoms,
cross-reactivity due to sequence homology orirrelevant recognition of cross-reactive epitopes [3].
And, in order to prescribe venom immunotherapy(VIT), determination of the sensitizing allergen is
crucial [34], especially if the patient had ananaphylactic reaction to only one insect. BAT brings
extra information when it comes to detecting thedominant allergen and the real source of venom
sensitization [35] and has the lowest rate of double
positivity of diagnostic tests for Hymenoptera allergy[36]. In the case of patients double-positive in BAT, theallergen that induces the highest degranulation isconsidered to be the primary sensitizing allergen [35].

BAT APPLICATION IN DRUG ALLERGY
In the context of an allergic reaction to drugs or related
compounds, several studies validate BAT for the
diagnosis of an IgE-mediated allergy fromneuromuscular blocking agents (NMBAs) [37], betalactam antibiotics and clavulanic acid as a blactamase inhibitor [38]–[40], iodinated radio-contrast
media (RCM) [41].
In case of small molecular weight drugs, called
haptens, which are not capable of FcεRI cross-linking
themselves (hapten concept [42]) a conjugation tocarrier molecules is required. The carriers are usually
proteins, for eliciting an immune reaction insusceptible individuals. Moreover, after the drug
metabolism, some reactive intermediates may beformed (pro-hapten concept; [43], [44]). Therefore, for
the investigation of drug hypersensitivity reactions theuse of drug metabolites and hapten-carrier conjugateshas been promoted [45]. For example, in the case of
propyphenazone (PP) hypersensitivity, basophils
reacted in BAT only after stimulation with the drugcarrier conjugate not when they were stimulated withpure PP [46]. Nevertheless, as a consequence oflacking knowledge regarding relevant determinants,
metabolic intermediates, their reactive functions, BAT
is most frequently performed with solutions of plain
drugs [46].
BAT presents some limitations listed in Table II
alongside with possible solutions. One of theselimitations is the stability of blood samples. Using
EDTA as anticoagulant means that the sample isstable only for a few hours, so a new method has beendeveloped – passive sensitization, but this method is45

applied only in research. The main steps of passive
sensitization are peripheral blood mononuclear cells(PBMC) isolation from a healthy donor, with wellcharacterized basophils; removal of IgE from thereceptor with lactic acid (pH 3.9), incubation withpatient serum (1 hour, 370 C) followed by a regular
BAT, as previously described. So the patient’s
samples are represented by serum, which can be
stored for a longer period at 40 C [7].

Table II. Limitations of basophil activation test and possible solutions [7], [47]Limitations
Reduced basophil reactivity overtime

Perform the basophil activation test
within max. 24h from blood collection.

Changes in temperature can affectbasophil reactivity

A good transportation system that
ensures a stable temperature of thesample.

Allergen exposure, chronic
inflammation and infection can
induce higher basophil reactivity
Immunosupressors, including oralcorticosteroids, may increase
basophil reactivity
Determining optimal allergenconcentration
Difficulties in establishing thethresholds for positivity

CONCLUSIONS
Basophil activation test is a functional test that mimics
an allergic reaction in vitro, proving to be a useful andsafe diagnostic method that can be used to evaluate
IgE-mediated hypersensitivity reactions. It can beconsidered a surrogate for provocation test and oral
food challenge. Currently, it is applied mostly inresearch because, in order to be accepted and usedin the clinical routine, it needs clinical validation. This

46

Possible solutions

Avoid basophil activation test after
allergen exposure or during infections.
Avoid immunosupressors before bloodcollection for basophil activation test.
A dose-response curve with patients andhealthy (exposed) controls for the
exclusion of unspecific stimulation.
Analysis of allergens in question withconfirmed allergic patients and healthy(exposed) controls instead of arbitrarily
chosen thresholds.
means standardised laboratory procedures,standardised allergen concentrations and optimalallergen positivity thresholds. Recent clinicalvalidations and standardisations made this test
available also for routine testing of several allergens.
Acknowledgements: This research was financed by
the INSPIRED (INovative Strategies for Prevention,
diagnosis and therapy of ragweed pollen Induced
REspiratory Diseases) project, COP 2014-2020
92/09.09.2016, P_37_747.

Fiziologia - Physiology • 2020.30.2(100)REFERENCES

15.

16.

1.
Ebo DG, Hagendorens MM, Bridts CH, Schuerwegh
AJ, De Clerck LS, Stevens WJ. In vitro allergy diagnosis:
should we follow the flow? Clin. Exp. Allergy, 2004; 34(3):17.
332-339.
2.
Hemmings O, Kwok M, McKendry R, SantosAF.
Basophil Activation Test: Old and New Applications inAllergy. Curr. Allergy Asthma Rep., 2018; 18(12): 77.
3.
Hoffmann HJ, et al. The clinical utility of basophil18.
activation testing in diagnosis and monitoring of allergic
disease. Allergy, 2015; 70(11): 1393-1405.
4.
Dvorak AM, MacGlashan DWJ, Morgan ES,
Lichtenstein LM. Vesicular transport of histamine in19.
stimulated human basophils. 1996.
5.
MacGlashan Jr DW. Basophil activation testing. J.
Allergy Clin. Immunol., 2013; 132(4): 777-787.
6.
McGowan EC, Saini S. Update on the performance20.
and application of basophil activation tests. Curr. Allergy
Asthma Rep., 2013; 13(1): 101-109.
7.
Hausmann OV, Gentinetta T, Bridts CH, Ebo DG. The
basophil activation test in immediate-type drug allergy.21.
Immunol. Allergy Clin. North Am., 2009; 29(3): 555-566.
8.
Eberlein B, et al. Optimizing of the basophil activation
test: Comparison of different basophil identification markers.
22.
Cytom. Part B Clin. Cytom., 2015; 88(3): 183-189.
9.
Hausmann OV, Gentinetta T, Fux M, Ducrest S,
Pichler WJ, Dahinden CA. Robust expression of CCR3 as asingle basophil selection marker in flow cytometry. Allergy,23.
2011; 66(1): 85-91.
10. Witting Christensen SK, Kortekaas Krohn I,
Thuraiaiyah J, Skjold T, Schmid JM, Hoffmann HJH.24.
Sequential allergen desensitization of basophils is non‐
specific and may involve p38 MAPK. Allergy, 2014; 69(10):25.
1343-49.
11. Santos AF, Shreffler WG. Road map for the clinicalapplication of the basophil activation test in food allergy. Clin.26.
Exp. Allergy, 2017; 47(9): 1115-24.
12. Monneret G. Is this time for CRTH2/DP2 in a flow
cytometric basophil activation test? Clin. Exp. allergy J. Br.27.
Soc. Allergy Clin. Immunol., 2008; 38(7): 1239.
13. Knol EF, Mul FPJ, Jansen H, Calafat J, Roos D.
Monitoring human basophil activation via CD63 monoclonal
antibody 435. J. Allergy Clin. Immunol., 1991; 88(3): 328-28.
338.
14. Sabato V, et al. Mechanism of phosphatidylserine
inhibition of IgE/FcεRI-dependent anaphylactic humanbasophil degranulation via CD300a. J. Allergy Clin.
Immunol., 2014; 134(3): 734-737.
Hennersdorf F, et al. Identification of CD13, CD107a, andCD164 as novel basophil-activation markers and dissectionof two response patterns in time kinetics of IgE-dependent
upregulation. Cell Res., 2005; 15(5): 325-335.

Fiziologia - Physiology • 2020.30.2(100)Suzukawa M, et al. IgE-and FcεRI-mediated enhancementof surface CD69 expression in basophils: role of low-levelstimulation. Int. Arch. Allergy Immunol., 2007; 143(suppl. 1):
56-59.
Ebo DG, Dombrecht EJ, Bridts CH, Aerts NE, De Clerck LS,
Stevens WJ. Combined analysis of intracellular signallingand immunophenotype of human peripheral blood basophilsby flow cytometry: a proof of concept. Clin. Exp. Allergy,
2007; 37(11): 1668-75.
Furuno T, Teshima R, Kitani S, Sawada J, NakanishiM.
Surface expression of CD63 antigen (AD1 antigen) in P815
mastocytoma cells by transfected IgE receptors. Biochem.
Biophys. Res. Commun., 1996; 219(3): 740-744.
MacGlashan Jr, D. Expression of CD203c and CD63 in
human basophils: relationship to differential regulation ofpiecemeal and anaphylactic degranulation processes. Clin.
Exp. Allergy, 2010; 40(9): 1365-77.
Bühring HJ, et al. The basophil activation marker defined by
antibody 97A6 is identical to the ectonucleotide
pyrophosphatase/phosphodiesterase 3. Blood, J. Am. Soc.
Hematol., 2001; 97(10): 3303-3305.
Hauswirth AW, et al. Interleukin-3 Promotes the Expressionof E-NPP3/CD203C on Human Blood Basophils in Healthy
Subjects and in Patients with Birch Pollen Allergy. Int. J.
Immunopathol. Pharmacol., 2007; 20(2): 267-278.
Santos AF, Lack G. Basophil activation test: food challenge
in a test tube or specialist research tool? Clin. Transl.
Allergy, 2016; 6(1): 1-9.
Santos AF, et al. Basophil activation test discriminates
between allergy and tolerance in peanut-sensitized children.
J. Allergy Clin. Immunol., 2014; 134(3): 645-652.
Thyagarajan A, et al. Evidence of pathway-specific basophil
anergy induced by peanut oral immunotherapy in peanutallergic children. Clin. Exp. Allergy, 2012; 42(8): 1197-1205.
Burks AW, et al. Oral immunotherapy for treatment of egg
allergy in children. N. Engl. J. Med., 2012; 367(3): 233-243.
Gómez E, et al. Role of the basophil activation test in the
diagnosis of local allergic rhinitis. J. Allergy Clin. Immunol.,
2013; 132(4): 975-976.
Glaumann S, Nopp A, Johansson SGO, Rudengren M,
Borres MP, Nilsson C. Basophil allergen thresholdsensitivity, CD‐sens, IgE‐sensitization and DBPCFC in
peanut‐sensitized children. Allergy, 2012; 67(2): 242-247.
Ceuppens JL, Bullens D, Kleinjans H, Van Der Werf J, Group
PBES. Immunotherapy with a modified birch pollen extract
in allergic rhinoconjunctivitis: clinical and immunologicaleffects. Clin. Exp. Allergy, 2009; vol. 39, no. 12, pp. 19031909, 2009.
29. Nopp A, Cardell LO, Johansson SGO, Öman H. CD‐
sens: a biological measure of immunological changes
stimulated by ASIT. Allergy, 2009; 64(5): 811-814.
30. Sturm GJ, Böhm E, Trummer M, Weiglhofer I,
Heinemann A, Aberer W. The CD63 basophil activation test

47

38.

in Hymenoptera venom allergy: a prospective study. Allergy,
2004; 59(10): 1110-17.
31. Eberlein‐König B, Varga R, Mempel M, Darsow U,39.
Behrendt H, Ring J. Comparison of basophil activation tests
using CD63 or CD203c expression in patients with insect
venom allergy. Allergy, 2006; 61(9): 1084-85.
32. Erdmann SM, Sachs B, Kwiecien R, Moll‐Slodowy S,40.
Sauer I, Merk HF. The basophil activation test in wasp
venom allergy: sensitivity, specificity and monitoring specific41.
immunotherapy. Allergy, 2004; 59(10): 1102-1109.
33. Korošec P, et al. Clinical routine utility of basophil
activation testing for diagnosis of hymenoptera-allergic
patients with emphasis on individuals with negative venomspecific IgE antibodies. Int. Arch. Allergy Immunol., 2013;42.
161(4): 363-368.
34. Eberlein-Konig B, Rakoski J, Behrendt H, Ring J. Useof CD63 expression as marker of in vitro basophil activation
in identifying the culprit insect venom allergy. J. Investig.43.
Allergol. Clin. Immunol, 2004; 14(1): 10-16.
35. Eberlein B, Krischan L, Darsow U, Ollert M, Ring J.
Double positivity to bee and wasp venom: Improved
diagnostic procedure by recombinant allergen–based IgE44.
testing and basophil activation test including data aboutcross-reactive carbohydrate determinants. J. Allergy Clin.
45.
Immunol, 2012; 130(1): 155-161.
36. Sturm GJ, et al. Inconsistent results of diagnostic tools
hamper the differentiation between bee and vespid venom
46.
allergy. PLoS One, 2011; 6(6): e20842.
37. Abuaf N, et al. Validation of a flow cytometric assay
detecting in vitro basophil activation for the diagnosis ofmuscle relaxant allergy. J. Allergy Clin. Immunol., 1999;47.
104(2): 411-418.
Eberlein B, Suarez IL, Darsow U, Rueff F, Behrendt H, Ring
J. A new basophil activation test using CD63 and CCR3 in48

allergy to antibiotics. Clin. Exp. Allergy, 2010; 40(3): 411418.
Sanz ML, et al. Flow cytometric basophil activation test by
detection of CD63 expression in patients with immediate‐type reactions to betalactam antibiotics. Clin. Exp. Allergy,
2002; 32(2): 277-286.
Torres MJ, et al. The diagnostic interpretation of basophil
activation test in immediate allergic reactions tobetalactams. Clin. Exp. Allergy, 2004; 34(11): 1768-75.
Pinnobphun P, Buranapraditkun S, Kampitak T, HirankarnN,
Klaewsongkram J. The diagnostic value of basophil
activation test in patients with an immediate hypersensitivity
reaction to radiocontrast media. Ann. Allergy, Asthma
Immunol., 2011; 106(5): 387-393.
Pichler WJ, Naisbitt DJ, Park BK. Immune pathomechanismof drug hypersensitivity reactions. J. Allergy Clin. Immunol.,
2011; 127(3): S74–S81.
Park BK, Pirmohamed M, Kitteringham NR. Role of drug
disposition in drug hypersensitivity: a chemical, molecular,
and clinical perspective. Chem. Res. Toxicol., 1998; 11(9):969-988.
Naisbitt DJ, Gordon SF, Pirmohamed M, Park BK.
Immunological principles of adverse drug reactions. Drug
Saf., 2000; 23(6): 483-507.
Himly M, et al. IgE-mediated immediate-type hypersensitivity
to the pyrazolone drug propyphenazone. J. Allergy Clin.
Immunol., 2003; 111(4): 882-888.
Steiner M, Harrer A, Himly M. Basophil reactivity as
biomarker in immediate drug hypersensitivity reactions—potential and limitations. Front. Pharmacol., 2016; 7: 171.
Santos A, Alpan O, Hoffmann HJ. Basophil activation test:
mechanisms and considerations for use in clinical trials andclinical practice. Authorea Prepr., 2020.

Fiziologia - Physiology • 2020.30.2(100)APLICAȚIILE CLINICE ALE TESTULUI DEACTIVARE A BAZOFILELOR ÎN DIAGNOSTICULBOLILOR ALERGICEREZUMATÎn prezent, diagnosticul alergiei mediate IgE se bazează în principal pe testul cutanat prick, dar această testarenu este recomandată pacienților care au avut anterior o reacție alergică severă la sursa alergenică testată.
Cuantificarea IgE seric specific are, de asemenea, neajunsurile sale. Prin urmare, testele celulare in vitro, cum arfi testul de activare a bazofilelor (BAT) sunt o bună alternativă de diagnostic. Testul de activare a bazofilelormăsoară hipersensibilitatea mediată IgE la diferiți alergeni fără a expune pacienții la un risc de anafilaxie. BATeste un test funcțional care măsoară expresia markerilor de activare precum CD63 și CD203c de pe suprafața
bazofilelor. Este utilizat pe scară largă în cercetare, dar validările clinice recente și standardizările au făcut
disponibil acest test și pentru testarea de rutină. În acest studiu, descriem mecanismele testului de activare abazofilelor și utilizarea clinică a acestuia în diagnosticul alergiilor alimentare, respiratorii, la venin și lamedicamente.
Cuvinte cheie: alergie, test de activare a bazofilelor, diagnostic alergie, CD63, CD203c

Fiziologia - Physiology • 2020.30.2(100)49

ASSESSMENT OF CELL CULTURE MEDIA VARIATIONINDUCING CYTOKINE SECRETION IN PBMCS, CD3+,
AND CD8+ IMMUNE CELLSOLTEANU GHEORGHE-EMILIAN1,2, CRISNIC DANIELA1,2,3, GRIJINCUMANUELA1, CRISTEAMIRABELA1, ZAMBORI CSILLA1,2,
IVAN
1,2
1,2
1,2
ALEXANDRA , BOJIN FLORINA , GAVRILIUC OANA , NEDEAEDUARD1,4, PAUNESCU VIRGIL1,2
1Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, Center for Gene and Cellular

Therapies in the Treatment of Cancer Timisoara – OncoGen; B-dul Liviu Rebreanu No. 156, RO300723, Timisoara, Romania
2 “Victor Babes” University of Medicine and Pharmacy Timisoara, Department of Functional
Sciences; Eftimie Murgu Square No. 2A, RO-300041, Timisoara, Romania
3 Biodim SRL, Ciprian Porumbescu Street, Timisoara, Romania
4 Biologos SRL, Curtea de Argeș, Romania
ABSTRACT
In vivo conditions, the innate immune arm signals shape the activation of naive T cells, however, it is unclear how
T cells maintain their function in vitro, in experimental settings, where different culture conditions signals influence
the effector T-cell function. In this study of two sets of healthy donors designated here group A (n=10) and group
B (n=10), we assessed the cytokine profile of PBMCs, cluster of differentiation 3 (CD3 +) cells and cluster ofdifferentiation 8 (CD8+) T cells, which were cultured in different environmental conditions, including Interleukin-2(IL-2) and/or CD3/CD28 T cell activator, supplemented culture media. The (IL-2), Interleukin-5 (IL-5) and Interferonγ (IFN-γ) secretion at 24, 48, 72, and 96 hours was determined in a 4.5-hour solid-phase ELISA immunoassay
method. The medium containing IL-2 and CD3/CD28 T cell activator was the one that induced increased secretionof IL-2, IL-5, and IFN-γ in all three cell populations, mainly in the CD3+ cells, irrespective of the time frame sampled.
Key words: PBMCs, cytokine, CD4+ T lymphocytes, CD8+ T lymphocytes, ELISAINTRODUCTION
This study was designed to provide a window inthe cytokine profile of peripheral blood mononuclear
cells (PBMCs), cluster of differentiation 3 (CD3+) cells,
and cluster of differentiation 8 (CD8+) T cells, grown in
different environmental conditions and to ascertain
which culture condition provided the best quantifiablesecretion of Interleukin-2 (IL-2), Interleukin-5 (IL-5),
and Interferon-γ (IFN-γ). All three cytokines of interestin this study are involved to a different degree invarious aspects of the immune response, from T cell
activation to regulatory induction [1-5]. Overall, thevast majority of evidence found in the literature credits
IL-2 with an essential part in the primary immune
response, a role in immune memory, induction ofregulatory T cells, and finally, IL-2 can also be
considered a survival factor for T cells via induction ofanti-apoptotic proteins [6-7].

In humans, IL-5 acts mainly on eosinophils andmast cells, and activation of B cells via dependent
pathways, therefore IL-5 can be viewed as achemokine bridge between the innate, and theacquired immune arms of the immune system [8-9].
IFN-γ is a soluble cytokine produced by a variety of
immune cells like CD4+ helper cells (Th1), CD8+cytotoxic T-lymphocytes (Tc1), non-cytotoxic innate
lymphoid cells (ILC), and predominantly by naturalkiller (NK), and natural killer T (NKT) cells as part ofthe innate immunological response (10). Interferon-γ
can be viewed as a central node in both the innate andadaptive immune response, with cellular effects likeupregulation of pathogen recognition, inhibition ofcellular proliferation, antigen processing andpresentation, antiviral, immunomodulation, andleukocyte trafficking [11-12].
Whole blood from two sets of healthy donors
designated here group A (n=10) and group B (n=10)

Received 5th of October 2020. Accepted 11th of November 2020. Address for correspondence: Olteanu Gheorghe-Emilian, “Victor
Babes” University of Medicine and Pharmacy Timisoara, Department of Functional Sciences, Eftimie Murgu Square No. 2A, RO300041, Timisoara, Romania; OncoGen-Centre for Gene and Cellular Therapies in the Treatment of Cancer, „Pius Brinzeu” Clinical
Emergency County Hospital Timisoara, Romania, Liviu Rebreanu No.156 Blv., Timisoara, RO-300723, Romania; phone/fax:+40356433282; e-mail: olteanugheorgheemilian@gmail.com

50

Fiziologia - Physiology • 2020.30.2(100)was used to provide Ficoll-Paque media density
gradient isolation of PBMCs, that were characterized
via a flow cytometry analysis. Using negative selection,
CD3+ and CD8+ T lymphocytes were isolated.
Afterwards, a flow cytometry analysis was performed
to view the distribution of cell populations. The cells ofinterest, PBMCs, CD3+ and CD8+ were plated into 24well plates, and grown using 3 types of environmental
conditions, including IL-2 and/or CD3/CD28 T cells
activator supplemented culture media.
Secretion of IL-2, IL-5, and IFN-γ was assessedwith the 4.5-hour solid-phase enzyme-linked
immunosorbent assay (ELISA) immunoassay method
on a windowed time frame analysis at 24, 48, 72, and96 hours [13-19]. All the samples were evaluated indouble and when necessary in triple pairs.
MATERIALS AND METHODS
Negative selection of human CD3+ T lymphocytes
The selection of human CD3+ T lymphocytes wasmade with the EasySep™ Human T Cell Enrichment
Kit (STEMCELLS Technologies, Vancouver, Canada)from frozen human PBMCs (two sets of healthy donors
group A (n=10) and group B (n=10)). This kit targets
non - T cells for removal with Tetrameric Antibody
Complexes and dextran-coated magnetic particles
recognizing specific cell surface markers. Unwanted
cells are labelled with antibodies and magnetic
particles and separated without columns using an
EasySep™ magnet. The cells of interest (CD3+ T
lymphocytes) are simply poured off into a new tube.
The cells were resuspended, 1.6 x 107 cells in 320 μl
buffer (EasySep™ Buffer, STEMCELLS Technologies,
Vancouver, Canada) into a polystyrene round-bottom
tube and incubated with 16μl of isolation cocktail(EasySep™ Human T Cell Enrichment Cocktail,
STEMCELLS Technologies) for 5 minutes at room
temperature. The magnetic particles 16μl/sample(EasySep™ D Magnetic Particles, STEMCELLSTechnologies) were added to the sample andincubated for 5 minutes at room temperature. The tube
was placed into the magnet and incubated for 5
minutes at room temperature, and the enriched cellsuspension was poured into a new tube. Following
isolation 3 x 106 CD3+ T lymphocytes/ml were obtained.
Negative selection of human CD8+ T lymphocytes
The selection of human CD8+ T lymphocytes wasmade with the EasySep™ Human CD8+ T Cell
Enrichment Kit (STEMCELLS Technologies) fromfrozen human PBMCs (two sets of healthy donors
group A (n=10) and group B (n=10)). This kit targets
non - CD8+ T cells for removal with Tetrameric
Fiziologia - Physiology • 2020.30.2(100)Antibody Complexes recognizing specific cell surface
markers and glycophorin A, and dextran-coatedmagnetic particles. Unwanted cells are labelled with
antibodies and magnetic particles and separated
without columns using an EasySep™ magnet(STEMCELLS Technologies). Following isolation, 3 x
106 CD8+ T lymphocytes/ml were obtained. Afterisolation PBMCs, CD3+ and CD8+ T cells were plated
into 24-well plates. All cell types were plated at 5 × 105
per well (1 ml/well), with three different types of media:
1. RPMI 1640 medium (Sigma-Aldrich Chemie GmbH,
Taufkirchen, Germany) supplemented with 5% humanserum (HS, Sigma-Aldrich) and 1% Penicillin –Streptomycin (Sigma-Aldrich); 2. RPMI 1640 medium(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
supplemented with 5% human serum (HS, SigmaAldrich) and 1% Penicillin – Streptomycin (SigmaAldrich) and 30 UI/ml IL-2; 3. RPMI 1640 medium(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
supplemented with 5% human serum (HS, SigmaAldrich) and 1% Penicillin – Streptomycin (SigmaAldrich), 30 UI/ml IL-2 (R&D Systems, Minneapolis,
USA), and CD3/CD28 Dynabeads (Dynabeads™
Human T-Activator CD3/CD28 for T Cell Expansion
and Activation, ThermoFisher Scientific, Waltham, MA
USA).
Flow cytometry
After using Ficoll-Paque media for density
gradient isolation of PBMCs, the resulting cells werealiquoted and stained with Simultest BD Biosciences(San Jose, California, USA) antibodies CD3 FITC/CD8
PE, afterwards following the instructions provided by
the manufacturer a two-colour flow cytometric analysis
was performed on the BD FACSVerse™ (San Jose,
California, USA) flow cytometer. Subsequently, after
the negative selection of human CD3+ T lymphocytes
from PBMCs, a Multitest BD 4 antibody cocktail(CD3/CD8/CD45/CD4) was used to highlight the
distribution of cell populations using the same BD
FACSVerse™ platform.
ELISA
The supernatant of all cell types, cultivated in thethree different media was collected at distinct time
points: 24h, 48h, 72h and 96h. The Quantikine®
Human Immunoassay (R&D Systems) was used forassessment of IL-2, IL-5, and IFN-γ secretion of thecells. The procedure followed the instructions provided
by the manufacturer. Shortly, the assay employs thequantitative sandwich enzyme immunoassay
technique, in which a monoclonal antibody specific forhuman cytokines is pre-coated onto a 96-wellpolystyrene microplate; standards and samples are
pipetted into the wells in duplicate, and IL-5, IL-2, or51

IFN-γ present in the sample is bound by theimmobilized antibody. The optical density wasdetermined using Tecan Infinite M200 Pro (Tecan,
Männedorf, Switzerland) microplate reader at 450 nm,

with wavelength correction at 570 nm, and eachcytokine concentration was calculated based on thestandard curve and regression equation.

Statistical analysis
Statistical analysis was performed using IBM, NY).

SPSS Statistics for Windows, Version 20.0. (IBM Corp.,
Armonk

Fig. 1. Graphical abstract of methods used in this study.

RESULTS
Flow cytometry analysis of PBMCs, CD3+, andCD8+ T lymphocytes
Flow cytometry of PBMCs from both sets ofhealthy donors, group A (n=10) and group B (n=10)

52

was analyzed with a gating strategy of CD45 (Ly-5),
CD3, CD4 with CD8, and CD8 with CD3, withpercentage representation of the cell population, bothsets of healthy donors showed similar results on theanalysis (Fig. 2).

Fiziologia - Physiology • 2020.30.2(100)Fig. 2. Gating strategy for lymphocytes population. A. Doted plot representing nucleated cells; B. Monocyte andlymphocyte population based on CD45 expression marker; C. cytotoxic T cells - CTLs (CD8+) and T cells (CD4+) fromtotal T cells; D. T cells (CD3+); E. CD8+ cells and percentage distribution of cellular populations.
Following the negative selection of CD3+ andCD8+ T lymphocytes from isolated PBMCs previously
quantified and analyzed, a flow cytometry analysis
was performed on the isolated CD3+ and CD8+ T
lymphocytes, the cell population was gated andpercentages were quantified for both sets of healthy
donors used in the study (Fig. 3).

Fig. 3. Flowcytometric analysis of gated selected
T cells (CD3+). A. Dot plot of total lymphocyte
population and CTLs (CD3+/CD8+); B. Percent valueof CTLs and T helper cells from the isolated T cells.
CD8+ T cells represented 35.95% of the total
CD3+ cells, while the CD4+ cells were 53.81%. We
obtained an enrichment of the total number T cells
from 67.89% to 87.27% after cultivation with activatorand expansion cocktail.

Fig. 4. Cells activated and expanded using
CD3/CD28. A. The Dynabeads can be seen coupledwith the cells. B. Digital magnification of T-cell
coupled with CD3/CD28 Dynabeads.
Solid-phase ELISA analysis of IL-2, IL-5, and IFN-γ
IL-2
Although IL-2 was added in the culture media
from the beginning, the IL-2 concentration determinedby ELISA method shows a time-dependent increase,similar to IL-5. The highest concentration value isreached after 96h by the CD3+ cell population, thus
confirming synergistic functioning of CD4+ and CD8+

Fiziologia - Physiology • 2020.30.2(100)cells when they are together in cell culture conditions.
PBMC whole population has a decreased ability tosecrete the mitogenic T cell factor. In Figure 5, thedifferences between cellular populations concerning
IL-2 secretion are well represented with statisticalsignificance bars.

53

Fig. 5. IL-2 concentration cell types per groups of patients. *p≤0.05, **p≤0.01, ***p≤0.001. Results from posthoc
MANOVA GTA Hochberg correction.

IL-5

ELISA assay performed at 24h, 48h, 72h, and96h revealed that IL-5 concentration increases in atime-dependent manner in all cellular population, forboth sets of healthy donors. However, the mostimportant increase was observed for CD3+ T cells, with54

a significant statistical difference compared with
PBMC, CD8+ or CD3- cellular populations.
Interestingly, the CD3- cellular populations secreted
large amounts of IL-5, probably due to monocytes andgranulocytes present in the cell culture. (p≤ 0.001
between PBMC and CD3-; Fig. 6)

Fiziologia - Physiology • 2020.30.2(100)Fig. 6. IL-5 concentration cell types per patient. *p≤0.05, **p≤0.01, ***p≤0.001. Results from posthoc MANOVAGTA Hochberg correction.

INF- γINF-γ, although normally secreted also by Tc1
CD8+ cells, it has the maximum concentration in the
supernatant of the CD3+ cells, where the contribution
is due to Th1 CD4+ cells (Fig. 6). Even though weused the same culture media for all cellular types,
supplemented with CD3/CD28 activator and IL-2, it

Fiziologia - Physiology • 2020.30.2(100)seems that the PBMC population is not able tocontribute with higher amounts of functional cytokines
compared to CD3+ population of cells. CD3+ cells havethe optimum cytokine secretion; further usage of thiscell population in further in vitro and in vivo studies ofinterleukin production is recommended.

55

Fig. 7. IFN-gamma concentration cell types per both sets of healthy donors. *p≤0.05, **p≤0.01, ***p≤0.001.
Results from posthoc MANOVA GTA Hochberg correction.

DISCUSSION
Scientific data credits IL-2 with a central role in T
regulatory (Treg) cell production and homeostasis,
with present studies attributing IL-2 a part in T memory
recall responses [6]. IL-5’s vital role in eosinophil
activation and generation is well established, thus, IL5 expands the innate immune response, and it
accentuates the acquired immune response linking thetwo main branches of the immune system [8]. IFN- γ
harmonizes many regulatory cellular programs via its
effects on transcriptionally relevant genes in the
immune response, specifically those that pertain tomacrophage activity [11]. To revenue the best output
of cytokines concerning stimulation of production andsecretion of cytokines, a well-established stimulatory
cocktail is used. In this study, the best outcome, orrevenue (quantifiable for cytokines secreted), is in theculture medium supplemented with CD3/CD28
Dynabeads, a stable, well-used activator andexpansion kit [20-22].

56

CONCLUSION
In our present study, there was no observablestatistical difference in cytotoxicity in the cell
populations in the first and second growth environment,
notable IL-2, IL-5, and IFN-γ secretions wereregistered in the third growth environment
supplemented with IL-2, and CD3/CD28 T cells
activator. The cell population with the most efficienteffector function was CD3+ irrespective of the timeframe sampled. Therefore, considering the findings in
our study and the evidence in the literature, the best
growth environment for the quantitative secretion of IL2, IL-5, and IFN-γ can be viewed as a cocktail between
normal growth media, IL-2, and CD3/CD28 T cells
activator supplementation.
Acknowledgement: This work was supported by the
grant “Chimeric Antigen Receptor Targeted
Oncoimmunotherapy with Natural Killer Cells (CARNK)”, POC 92/09/09/2016, ID: P_37_786, MySMIS
code: 103662.

Fiziologia - Physiology • 2020.30.2(100)REFERENCES
1.
2.

3.

4.

5.
6.
7.
8.

9.
10.

11.
12.

13.

Hay FC. Chapter 10 – The cytokines; Practical
Immunology Frank C. Hay, Olwyn M.R.
Westwood & Paul N. Nelson, 2002, page 301
Murphy K, Weaver C. Chapter 9 – Chapter 9: Tcell-MediatedImmunity
Janeway's
immunobiology. Garland Science; 2016 Mar 1,
page 346.
Gao GF, Jakobsen BK. Molecular interactions ofcoreceptor CD8 and MHC class I: the molecularbasis for functional coordination with the T-cell
receptor. Immunol Today. 2000; 21(12):630-6.
Leahy DJ, Axel R, Hendrickson WA. Crystal
structure of a soluble form of the human T cell
coreceptor CD8 at 2.6 Å resolution. Cell. 1992;
68(6):1145-62.
Malek TR. The biology of interleukin-2. Annu. Rev.
Immunol. 2008; 26:453-79.
Ross SH, Cantrell DA. Signaling and function ofinterleukin-2 in T lymphocytes. Annu. Rev.
Immunol. 2018; 36:411-33.
Takatsu K, Kouro T, Nagai Y. Interleukin 5 in thelink between the innate and acquired immune
response. Adv Immunol. 2009; 101:191-236.
Rasmussen R, Takatsu K, Harada N, Takahashi
T, Bottomly K. T cell-dependent haptenspecificand polyclonal B cell responses require releaseof interleukin 5. J Immunol. 1988; 140(3):705-12.
Bonilla FA, Oettgen HC. Adaptive immunity. J
Allergy Clin Immunol. 2010; 125(2):S33-40.
Schroder K, Hertzog PJ, Ravasi T, Hume DA.
Interferon‐ γ: an overview of signals,
mechanisms and functions. J Leukoc Biol. 2004;
75(2):163-89.
Boehm U, Klamp T, Groot M, Howard JC. Cellularresponses to interferon-γ. Annu Rev Immunol.
1997; 15(1):749-95.
Levring TB, Hansen AK, Nielsen BL, Kongsbak
M, Von Essen MR, et al. Activated human CD4+
T cells express transporters for both cysteine andcystine. Sci Rep. 2012; 2:266.
De Jager W, te Velthuis H, Prakken BJ, Kuis W,
Rijkers GT. Simultaneous detection of 15 human

Fiziologia - Physiology • 2020.30.2(100)

14.

15.

16.

17.
18.
19.

20.

21.

22.

23.

cytokines in a single sample of stimulated
peripheral blood mononuclear cells. Clin. Diagn.
Lab. Immunol. 2003; 10(1):133-9.
Hennig C, Rink L, Fagin U, Jabs WJ, Kirchner H.
The influence of naturally occurring heterophilic
anti-immunoglobulin antibodies on direct
measurement of serum proteins using sandwich
ELISAs. J Immunol Methods. 2000; 235(1-2):7180.
Nahar P. Enzyme-linked immunosorbent assay
procedure at higher temperature enhances
speed of the assay. Community Contributed
Protocol Exchange. 2013; 9:12.
Aghaeepour N, Finak G, Hoos H, Mosmann TR,
Brinkman R, Gottardo R, et al. FlowCAP
Consortium, Dream Consortium. Criticalassessment of automated flow cytometry data
analysis techniques. Nat Methods. 2013;10(3):228.
Lefkovits I, Pernis B, editors. Immunologicalmethods. Elsevier; 2014 May 12.
Katz F. Methods in immunology: A laboratory text
for instruction and research by Justine S.
Garvey, Natalie, E. Cremer and Dieter H.
Sussdorf. Pp 545. WA Benjamin Inc., Reading,
Mass. Advanced Book Program, 1977.
Biochemical Education. 1978; 6(3):65.
Imada M, Simons FE, Jay FT, HayGlass KT.
Antigen mediated and polyclonal stimulation ofhuman cytokine production elicit qualitatively
different patterns of cytokine gene expression.
International immunology. 1995; 7(2):229-37.
Nguyen DH, Hurtado-Ziola N, Gagneux P, Varki
A. Loss of Siglec expression on T lymphocytes
during human evolution. Proc Natl Acad Sci U S
A. 2006; 103(20):7765-70.
Larrea E, Riezu-Boj JI, Gil-Guerrero L, Casares
N, Aldabe R, et al. Upregulation of indoleamine 2,
3-dioxygenase in hepatitis C virus infection. J
Virol. 2007; 81(7):3662-6.
Schade AE, Schieven GL, Townsend R,
Jankowska AM, Susulic V, et al. A small-molecule
protein tyrosine kinase inhibitor, inhibits T-cell
activation and proliferation. Blood. 2008;
111(3):1366-77.

57

EVALUAREA VARIAȚIEI MEDIULUI DE CULTURĂASUPRA SECREȚIEI DE CITOKINE A PBMCS,
LIMFOCITE T CD4+ ȘI CD8+
REZUMATÎn condiții in vivo, sistemul imun înnăscut modulează activarea limfocitelor T naïve, dar este încă neclară
modalitatea prin care limfocitele T reușesc să își mențină funcția in vitro, în condiții experimentale, careinfluențează funcția limfocitelor T efectoare. În acest studiu, pe două seturi de donatori sănătoși, denumiți ca grup
A (n=10) și grup B (n=10), am testat profilul citokinic al PBMCs, limfocitelor T CD3+ și al limfocitelor T CD8+, careau fost cultivate în medii de cultură diferite, care au inclus suplimentarea cu IL-2 și/sau activator al limfocitelor T,
CD3/CD28. A fost determinată secreția citokinelor de tip IL-2, IL-5 și -γ (IFN-γ) la 24, 48, 72 și 96 de ore, printestări assay-uri imune de tip ELISA. Mediul de cultură conținând IL-2 și activator limfocitar CD3/CD28 a fost cel
care a dus la secreția crescută de IL-2, IL-5 și IFN-γ în toate cele 3 populații celulare investigate, mai ales la nivelul
limfocitelor T CD3+, indiferent de intervalul de timp la care a fost investigată.
Cuvinte cheie: PBMCs, citokine, limfocite T CD4+, limfocite T CD8+, ELISA

58

Fiziologia - Physiology • 2020.30.2(100)THE USE OF SPECIFIC ASSAYS TO QUANTIFYOXIDATIVE STRESS IN MULTIDRUG-RESISTANTBACTERIACARMEN TATU1,2, CRISTINA GASPAR1, MARIUS GEORGESCU2, ALINAGEORGIANA SIMINA1, GABRIELA TĂNASIE1,2*, FLORINA BOJIN1,2,
DANIELA PUȘCAȘIU3, CARMEN PANAITESCU1,2
1OncoGen Center, Clinical County Hospital Timișoara
2Immuno-Physiology and Biotechnology Center, Department III of Functional Sciences, University of Medicine and Pharmacy VictorBabeș Timișoara
3Microscopic Morphology Department, Cellular and Molecular Biology Discipline, University of Medicine and Pharmacy Victor Babeș
Timișoara

ABSTRACTDuring oxidative stress the balance between the production of reactive oxygen species and antioxidant protective
systems is disturbed. Reactive oxygen species (chemical species with an odd electron) include: hydroxyl radical(OH-), superoxide anion (O2˙‾), hydrogen peroxide (H2O2), while antioxidant protective systems are classified into:
enzymatic antioxidants (superoxidismutase, glutathione peroxidase, glutathione S-transferase) and non-enzymatic
antioxidants (reduced glutathione, vitamin E, ascorbic acid, selenium, beta-carotene and vitamin A). In the face of
microbial invasion, cells defend themselves by releasing reactive oxygen species, but almost all bacteria have
evolved and are able to form and use a range of antioxidants to manage the toxic effects of oxidative stress. To
understand the defense mechanisms of bacteria, bacterial models were used that managed to characterize theiradaptive responses. The development of methods capable of correlating oxidative stress with the bacterial
response could contribute to obtaining a perspective on the development of new antibacterial strategies.
Keywords: oxidative stress, reactive oxygen species, enzymatic antioxidants, non-enzymatic antioxidants

INTRODUCTION
Oxidative stress is defined as the situation inwhich there exists an imbalance between the
production of reactive oxygen species (ROS) andreactive nitrogen species (RNS) and antioxidant
defense [1]. Reactive oxygen species are produced byliving organisms as a result of normal cellular
metabolism and of environmental factors - such as air
pollutants or cigarette smoke. At low to moderateconcentrations, they work in physiological cellular
processes, however at high concentrations, they areable to damage cellular structures such ascarbohydrates, nucleic acids, lipids, proteins, and, in
addition, they can alter their functions. Aerobic

organisms have developed two systems for controlling
reactive oxygen species: the first and most important the prevention of their formation and the second - theirneutralization as soon as they form. Integrated
antioxidant systems include enzymatic and nonenzymatic antioxidants that are usually effective inblocking the harmful effects of ROS, but under certain
conditions, the balance between oxidants andantioxidants is disturbed in favor of oxidants. This
oxidants/antioxidants imbalance is the definition ofoxidative stress [2]. Although ROS are cell defense
systems against microbial invasion, it has been shownthat almost all bacteria have evolved and are able touse an arsenal of adaptive and inducible antioxidants,
so that they can manage the toxic effects of ROS [4].

Received 12th of October 2020. Accepted 11th of November 2020. Address for correspondence: Prof. Univ. Dr. Tanasie Gabriela,
Immuno-Physiology and Biotechnology Center, Department of Functional Sciences, „Victor Babes” University of Medicine andPharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Romania; OncoGen - Centre for Gene and Cellular Therapies in
the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency County Hospital Timisoara, Romania, Liviu Rebreanu No.156 Blv.,
Timisoara, RO-300723, Romania; phone/fax: +40724250433 / +40356433282; e-mail: gtanasie@umft.roFiziologia - Physiology • 2020.30.2(100)59Bacterial cells have been shown to possess a numberof proteins that are able to repair oxidized proteins
resulting from ROS attack [3].

REACTIVE OXYGEN SPECIES (ROS) arechemical species that have an odd number ofelectrons. Being highly reactive molecular fragments,
free radicals react with organic molecules,
macromolecules and membranes, disrupting
intracellular homeostasis and damaging subcellular
structures (mitochondria and endoplasmic reticulum)
and their decomposition products enter the structureof lipofuscin pigments (also called wear and tearpigments) or of age pigments. They are produced in
the cell, either accidentally or during phagocytosis.
ROS occur in certain redox reactions in which changes
occur, as a result of which the substance oftenchanges its biological function (becomes morehydrosoluble or takes part in another chain of
metabolic reactions). ROS intervene as the main
amplification factor in the production of "oxidative
lesions": peroxides, hydroperoxides, endoperoxides
and epoxides, to which a new amplification resulting
from the oxidants/antioxidants imbalance is added:
through excess of pro-oxidants, doubled by lack ofantioxidants. The amplification and accumulation overtime of free radicals and oxidative stress (chronic
stress), expressed by the imbalance of the ratio ofexcess pro-oxidants/deficiency of antioxidants,
causes the shift of the anabolism/catabolism balance
towards anabolic stress (hypo-anabolism)/catabolic
stress(hyper-catabolism)withintracellularaccumulation of insoluble waste. Oxidative damage
can have a devastating effect on the structure andactivity of proteins and can even lead to cell death.
Amino acids which contain cysteine and methionine
sulfur are particularly sensitive to reactive oxygen
species and reactive chlorine species, which candamage proteins [3].

Types of ROS produced in vivo
1. The hydroxyl radical – OH- is one of the mostreactive free radicals, it appears by exposing the bodyto ionizing radiation. It can react with almost any
molecule of the living cell; once formed it destroys
everything around it, but it cannot migrate long
distances into the cell. Almost all the destructive
effects caused by excessive exposure to ionizing
radiation are initiated by the attack of the hydroxyl
radical on proteins, carbohydrates, DNA, lipids [6].
2. The superoxide anion - O2˙‾ is produced byphagocytes (neutrophils, eosinophils, monocytes,
macrophages), as well as other cells such as
60

lymphocytes, fibroblasts, vascular endothelial cells inorder to inactivate viruses and bacteria. O2˙‾ isthought to be involved in the transmission of theintracellular signal as well as in the regulation of cell
growth. The superoxide anion, as well as hydrogen
peroxide, can be generated by autooxidation reactions
in which compounds such as catecholamines,
tetrahydrofolate react with oxygen to form thesuperoxide [6]. It will oxidize other compounds
initiating reaction chains with the formation of otherfree radicals. The intimate mechanism of formation
involves the addition of an electron to molecularoxygen. This process is mediated by nicotine adenine
dinucleotide phosphate [NAD(P)H] oxidase or
xanthine oxidase or by a mitochondrial electrontransport system. The main site for the production of
the superoxide anion is the mitochondria, that is thatpart of the cell specialized in the production of
adenosine triphosphate. Normally, electrons aretransferred through the mitochondrial electrontransport chain to reduce oxygen to water, but about 1
to 3% of all electrons leak out of the system andproduce superoxide. NAD(P)H oxidase is found inpolymorphonuclear leukocytes, monocytes andmacrophages. During phagocytosis, these cells
produce an explosion of superoxide leading tobactericidal activity. Superoxide is converted tohydrogen peroxide by the action of superoxide
dismutase (SOD). The superoxide anion is capable ofcausing a range of damaging effects, including
destruction of endothelial cells with increased
microvascularpermeability,
formationofchemoattractants such as leukotriene B4, recruitmentof neutrophils for inflammation, lipid peroxidation,
destruction of DNA, and formation of the peroxynitrite
anion (ONOOˉ). which in its turn can cause a numberof changes [5].
3. Hydrogen peroxide (H2O2) occurs in natural
habitats through reactions between sulfur and oxygenat the oxic/anoxic interfaces, by photochemical
reduction of oxygen by chromophores or the redox
cycle of pigments. In addition, plants and animals can
excrete H2O2 to destroy microbes [7]. H2O2 can beconsidered an ideal weapon because it is small andnot electrically charged. These characteristics
facilitate its passive crossing of the cell membrane at
speeds similar to those of water [8]. Once inside thecytoplasm, it disrupts the iron metabolism, virtuallydestroying the cell. An experimental model using the
Escherichia Coli bacterium was used to identify themechanisms by which H2O2 affects cell survival. The
results showed that exogenous H2O2 damages thecell's DNA and is therefore mutagenic. This effect isknown as the Fenton reaction, in which H2O2 reacts
with intracellular iron [9].
Fiziologia - Physiology • 2020.30.2(100)The explanation why H2O2 is so harmful to cells when
it interferes with the iron metabolism comes with thediscovery that it inactivates two families of ironcontaining enzymes: non-redox mononuclear
enzymes and dehydrases [10, 11]. Basically, H2O2
oxidizes iron from these enzymes and suppresses
their activity. It has been observed that only 0.5 μM
H2O2 is sufficient to destroy the cells in this manner. It
has also been observed that following the Fenton
reaction, a series of lesions appear in the cell that cancause mutagenesis [10].
ANTIOXIDANT PROTECTIVE SYSTEMS
The increase in the O2 concentration of the
Earth's atmosphere 3 billion years ago forced theprimitive forms of life to adapt by creating antioxidant
protective systems. These unicellular algae andbacteria not only adapted to the increasing
concentration of O2 but begun to use it as a promoterof the evolution of life and initiator of many essential
processes [12]. The body defends itself against
excess free radicals with the help of non-enzymatic(natural and synthetic) or enzymatic antioxidants. To
increase efficiency, enzymatic and non-enzymatic
antioxidants located in various environments membrane, cytoplasm, extracellular fluid, and inseveral subcellular fractions - act on the same free
radical species [6].

Enzymatic antioxidants
1. Superoxidismutase (SOD) - is found in any
aerobic cell, in the mitochondria and cytosol. It
converts the superoxide anion to hydrogen peroxide[13]:
2O2˙ˉ + 2H+  H2O2 + O2
There are several forms of SOD: Fe-SOD in lower
organisms and in higher organisms Cu-Zn-SOD in thecytoplasm and Mn-SOD in the mitochondria. Although
SOD is found quite abundantly in mammalian cells,
diseases have been observed in which the activity ofthe enzyme is low, either due to the presence of
inhibitors, or due to reduced biosynthesis. Decreased
SOD has been found in certain foci: inflammation,
tumors, cataracts and ischemia of large organs (heart,brain). Attempts have been made to introduce purified
SOD in the treatment of diseases, with variable results[14]. Outstanding results were obtained with synthetic
SOD, but the high toxicity of this product limits its
clinical use.

Fiziologia - Physiology • 2020.30.2(100)

2. Catalase is found in all aerobic cells, in
mitochondria and peroxisomes. It acts on hydrogen
peroxide molecules, with a detoxifying effect:
H2O2 + H2O2  O2 + 2H2O
The large amount of catalase present in red blood
cells and liver seems to justify the theory of its
involvement in processes where high amounts of H2O2
are produced [15].
3. Glutathione peroxidase contains selenium as its
active part. The accumulation of the oxidized form ofglutathione, formed both through the action of GSH
peroxidase or non-enzymatically, seems to be anindicator of oxidative stress at the cellular level. GSH
peroxidase also acts as a regulator in prostaglandinbiosynthesis by inhibiting lipoxygenase. The
importance of GSH peroxidase in prostaglandinbiosynthesis is so great that in selenium deficiency
endothelial lesions of some vessels and platelet
aggregation disorders occur, due to the impairment of
prostacyclines biosynthesis [6].
4. Glutathione S-transferase is found intracellularly.
It catalyzes the conjugation of glutathione withdifferent compounds under conditions of chemical
pollution [6]. A wide variety of isoenzymes of thisantioxidant exist, that are ubiquitously expressed inhumans. In addition to their catalytic role inconjugating GSH to a variety of harmful components,
several isoenzymes reduce hydroperoxides anddetoxify the end products resulting from lipid
peroxidation.

Non-enzymatic antioxidants

Unlike enzymatic antioxidant systems, which are
limited to the four enzymes, non-enzymatic
antioxidants comprise a very large group of
substances whose list is constantly growing.
1. Reduced glutathione (L-alpha-glutamyl-Lcysteinyl-glycol) (GSH) is the main intracellular nonenzymatic antioxidant. It is a carrier of free -SHgroups. Both non-enzymatically and acting as acoenzyme of GSH peroxidase or transferase, GSH is
involved in the neutralization of endogenous orenvironmental harmful substances, of free oxygen
radicals generated by acute and chronic intoxications
and in reactions with organic compounds and free
radicals [6].
2. Vitamin E (α-tocopherol) is recognized as being
the most important liposoluble antioxidant, whosebasic role is to maintain the integrity of membranes.
Vitamin E is a target for free radicals and is an effective
inhibitor of the evolution of the oxidative chain [16].
61Vitamin E has been shown to regenerate continuously,
and this increases its effectiveness in pathologicalconditions in which cell lysis occurs [17].
3. Ascorbic acid. Discussions on the therapeuticutility of vitamin C have had a great echo with thegeneral public. The main involvements of vitamin C
are the anti-infective role based on the modulating
effects of ascorbic acid in cellular immunity, thedetoxifying role based on accelerating the metabolism
of xenobiotics, the antioxidant role – its mostcontroversial since a number of papers list vitamin C
among free radicals, while other papers argue in favorof its pro-oxidant role, especially with in vitro
experiments [18].
4. Selenium acts not only by GSH peroxidase, butalso independently or in association with vitamines[19].
5. Beta-carotenes and vitamin A, while having
strong antioxidant effects, are known especially wellamong anticancer compounds. Numerous works havedemonstrated their antioxidant character, peroxidation
propagation blocker, and singlet oxygen scavenger[6].

OXYGEN REACTIVE SPECIES IN BACTERIAOxidative stress in microbial cells shares many
similarities with other cell types, but has its specificcharacteristics that may differ between prokaryotic andeukaryotic cells [22]. A series of studies aimed at
elucidating the mechanism by which bacteria react to
ROS attack, used bacterial models (Escherichia Coli
and Pseudomonas aeruginosa), thus succeeding incharacterizing their adaptive responses, resulting inthree conclusions [4, 20]:
• Firstly, each species of ROS triggers its own
type of response;• Secondly, the ROS response is underregulatory control in terms of inducing
proteins and enzymes with a role inantioxidant defense;• Thirdly, the response mechanisms are
relatively preserved in most bacterial species.
Therefore, it appears that the results obtainedusing the Escherichia Coli and Pseudomonas
aeruginosa models can be extrapolated tovisualize the general bacterial responses to
ROS. Both bacterial species seem to react inthe same way: they achieve antioxidant
defense by acting at all possible levels [4]:•bacteria are constantly trying toprevent ROS attacks;
62

once the ROS offensive is launched,attempts are made to intercept andblock the radicals;•
if damage has been caused, repairs
are made.
Thus, bacterial cells prepare their potential
defense by maintaining a basal level of enzymes
capable of eliminating ROS such as O2- and H2O2, andthus keeping their concentration within safety limits.
The defense against ROS is also performed byantioxidant enzymes, metals, radical scavengers,
protective proteins and repair enzymes [21]. Given the
recent increase in antibiotic resistance, certain
antimicrobial strategies based on the generation of
ROS may be considered. To elaborate on the way this
could be achieved, we will present a brief review of
how antibiotics act on bacteria [23, 24]. It is not yet
clear whether this is achieved through a single
mechanism or multiple ones, which once triggered will
eventually lead to the death of the bacterium. What iscertain is the fact is that the main antibacterial drugs
have three targets in bacteria:• Bacterial cell wall
Penicillin,forexample,promotestranspeptidation, that is inhibiting theformation of peptidoglycan-type cross-links inthe bacterial cell wall. The result is a veryfragile cell wall that disintegrates, killing thebacteria.
• Bacterial synthesis of proteins
Antibiotics can inhibit the synthesis of certain
proteins. Tetracycline, for example, can crossthe bacterial wall membrane and accumulate
in high concentrations in its cytoplasm. It thenattaches to the 30S ribosomal subunit andblocks RNA and thus stops the elongation of
the protein chain [23, 25].
• Replication and repair of bacterial DNA
Bacterial
chromosomal
topologyismaintained through the activity of three
enzymes: topoisomerase I, topoisomerase IV
and DNA gyrase (topoisomerase II). The
action of these enzymes is disorganized bysynthetic quinolone, an antibiotic that targets
DNA-topoisomerase complexes. This action
leads to complete inhibition of cell division,
with bacteriostatic effect and eventually thedeath of the bacterium [25].
A study conducted by Kohanski and colleagues
in 2007 showed that three of the main classes ofbactericidal drugs use a common inactivation
mechanism that stimulates the production of lethal
doses of hydroxyl radicals by the Fenton reaction [26].
Triggered oxidative stress contributes to antibioticmediated cell death. Also in this study it was shownthat oxidative stress is involved in acquiring antibiotic•

Fiziologia - Physiology • 2020.30.2(100)resistance of pathogenic bacteria by altering theantioxidant defense mechanisms of cells. In
conclusion, oxidative stress can be considered as apossible mechanism for antibiotics’ action, and, on theother hand, as a factor involved in the development ofbacterial resistance to antibiotics [27].

could contribute to obtaining a perspective on thedevelopment of new antibacterial strategies.
Unfortunately, the ROS's tendency to acquire
electrons makes them very reactive, with a shortlifespan and therefore very difficult to detect. However,a number of methods useful in this regard do exist.
1. Methods based on chemiluminescence

PROTECTIVE MECHANISMS OF BACTERIAAGAINST OXIDATIVE STRESS
Bacteria live in a toxic world in which theircompetitors excrete hydrogen peroxide or redox
compounds that generate superoxide. To cope with
the ROS attack, bacteria developed a series ofdefense strategies. The most frequently used study
model is with E. Coli and the enzymes involved are
SOD in the study of superoxide anion degradation andperoxidases and catalases for the degradation ofhydrogen peroxide. Mutants that lack these enzymes
also suffer from alterations of enzymes that have iron
as a cofactor (catalases, peroxidases andribonucleotide reductases). Also, following thereaction between intracellular bivalent iron andhydrogen peroxide, hydroxyl radicals result, which inturn will react with all biomolecules. At the level ofDNA, these radicals produce lesions that areincompatible with the activity of DNA polymerase andwith replication, and if the lesions are major, the end
result is cell death.The results of the study publishedby Imlay JA in 2015 suggest that bacteria protect
themselves by activating so-called regulons controlled
by a number of transcription factors [11]. A key
objective of the current activity is to identify the natural
circumstances in which regulons are activated. They
perceive peroxide when they oxidize key portions of
thiolate or iron, they then induce the formation of setsof proteins that will trigger the defense of vulnerable
enzymes. In some non-enteric bacteria, some of thetranscription factors control the synthesis and exportof redox cycle compounds, while in enteric bacteria thecell defends against the same agents. In addition,some bacteria can induce enzymes that defend thecell against the superoxide produced by thesecompounds. The results of studies on non-entericbacteria are promising and seem to provide someclarity in the future on how microbes protect
themselves against the attack of ROS.

QUANTIFICATION OF OXIDATIVE STRESS IN
PROKARYOTIC SYSTEMS (BACTERIA)

Several studies have used chemiluminescence totest for the presence of ROS in bacteria. The principleof the method is to bind the dye/enzyme complex(luminophores) to the target reactive oxygen species(ROS) and to emit light, the intensity of which is directly
proportional to the ROS. The measurement is madewith luminometers, and the values are expressed inrelative units of light (RLU) [28] or by flow cytometry[29].
Albesa and colleagues used chemiluminescence
to assess the involvement of oxidative stress,
especially the role of superoxide in the action of
antibiotics against various bacteria, including
Staphylococcus
aureus,Escherichia
coli,
Pseudomonas aeruginosa and Enterococcus faecalis[30]. Using chemiluminescence, this group of
researchers found that various antibiotics can increase
the release of superoxide in different strains, but only
those antibiotic-sensitive strains respond to oxidative
stress. Other studies have shown the role of hydroxyl
radicals (OH•) as an essential factor in the responseto oxidative stress. A study by Kohanski et al. tracked
E. coli treated with norfloxacin, ampicillin andkanamycin and Staphylococcus aureus treated with
norfloxacin and chloramphenicol and demonstrated
the release of hydroxyl radicals by the Fenton reaction[26]. A specific dye - hydroxyphenyl fluorescein (HPF)
was used to evaluate the formation of hydroxyl
radicals. Similar results were observed with
norfloxacin-treated E. coli after HPF addition. A groupof researchers coordinated by Yeom J demonstrated
in 2010 that antibiotics could accelerate cell death bypromoting the Fenton reaction that leads to anoxidative stress response in ampicillin-treatedPseudomonas aeruginosa [29]. The results of thestudy show that the antibiotic action is affected bymodulating the reduction of nicotinamide-adenine
dinucleotide (NADH) levels and iron chelation. In
addition to direct or indirect measurements of ROS,
the involvement of ROS in cell death and antibiotic
resistance can also be assessed through the use of
ROS scavengers.

The development of methods capable ofcorrelating oxidative stress with the bacterial response
Fiziologia - Physiology • 2020.30.2(100)632. Methods which use flow cytometry
This type of method is relatively new inquantifying oxidative stress in antibiotic-treatedbacteria. In 2018, a group of researchers from the
University of Geneva led by Daniel Manoil set out toevaluate the oxidative stress in bacteria using flow
cytometry [31]. They used the DeepRO CellROX® kit
which combines an ROS-sensitive fluorophore(CellROX® Deep Red) and a dye for assessing cell
viability (SYTOX® Blue) to allow the detection of nonoxidized, oxidized and damaged cells. The bacteriaEnterococcus faecalis and Fusobacterium nucleatum
were studied and subjected to oxidative stress. An
optimal concentration of CellROX® was determined
on Enterococcus faecalis and Fusobacterium
nucleatum exposed to oxidative stress (H2O2).
Bacteria were exposed to different concentrations of
H2O2 and labeled with CellROX® to check if
fluorescence increased with oxidative stress. Bacteria
exposed to H2O2 were also double stained with
CellROX® and SYTOX® Blue and analyzed by flow
cytometry. Both strains were labeled with CellROX®
Deep Red. The fluorescence of CellROX® Deep Redlabeled bacteria increased accordingly with oxidative
stress. Flow cytometry analysis of double-stained
samples showed subpopulations of bacteria withincreased CellROX® signal when stressed and ahigher absorption of SYTOX® Blue under higher
oxidative stress. The results of this study indicate that
CellROX® Deep Red can be applied to measureoxidative stress in E. faecalis and F. Nucleatum andthe combination of CellROX® and SYTOX® Blue
allowed the discrimination of non-oxidized, oxidized
and damaged bacteria.
3. Methods based on the use of free radical
scavengers/antioxidants
The principle of this method is based on the
addition of antioxidants (thiourea; 2,2-bipyridyl;
glutathione; ascorbic acid) in bacterial cultures
exposed to antibiotics that cause oxidative stress that
protects cells from the harmful effects of ROS and thusincreases the survival rate of bacteria. Goswami andcolleagues investigated the involvement of ROS in theaction of Ciprofloxacin on Escherichia coli [32].
Ciprofloxacin is an important and frequently usedmember of the fluoroquinolones family of drugs. Its
mechanism of action is based on the inhibition of theactivities of DNA topoisomerase II and DNA
topoisomerase IV respectively, eventually leading to
the death of bacterial cells. In addition, an increase in
ROS in bacterial cells in response to the action of
ciprofloxacin has been demonstrated. These
researchers investigated the role of ROS in the64

antibacterial action of ciprofloxacin by studying theeffects of various antioxidant compounds on thesusceptibility of Escherichia coli to ciprofloxacin.
Among the antioxidants tested, glutathione andascorbic acid provided substantial protection against
ciprofloxacin. The involvement of the superoxide anion(O2) and hydrogen peroxide (H2O2) in the antibacterial
action of ciprofloxacin was analyzed using superoxide
dismutase, catalase and alkyl hydroperoxide
reductase. The results of this study suggest that O2and H2O2 may be involved in the antibacterial action of
ciprofloxacin, that ROS species may have a similarrole in the antibacterial action of all fluoroquinolones,
and that glutathione-mediated protection is not a
general phenomenon but one that is specific tofluoroquinolones.
4. Methods based on the use of mutant strains
Investigating the processes related to oxidative
stress and the role of ROS in bacteria exposed to
antibiotics provides valuable information on themechanism of cell death mediated by bactericidal
antibiotics and the involvement of ROS in this process.
Another strategy for evaluating the release of ROS in
antibiotic-treated bacteria is the use of mutant strainsthat have a modified antioxidant defense mechanism.
The antioxidant defensive system of bacteria includes
specific antioxidant enzymes, including superoxide
dismutase (SOD), catalase and peroxidase. By
manipulating E. coli strains, a knockout strain can begenerated to create a state of artificial imbalance thatallows the selective study of oxidant/antioxidant
mechanisms.
The principle of this determination method isbased on:
1. use of antibiotic-sensitive strains
compared to resistant or knockout strains(bacteria whose oxidative stress defense
genes, in particular those responsible for
SOD, catalase and peroxidase, have been
inactivated through genetic manipulation)
2. highlighting certain genes that are
expressed in response to oxidative stress
5. Proteomic investigations
Are based on the use of the following methods:• Mass spectrometry - ESI (electrosprayionization) and MALDI (ionization by laser
desorption assisted by a solid matrix) –identification of proteins/isoforms affected byoxidative stress and their grouping according
to the function they perform

Fiziologia - Physiology • 2020.30.2(100)Gel electrophoresis - identification ofproteins/isoforms affected by oxidative
stress, based on molecular weight.
• PCR - identification and subsequent
expression of DNA sequences responsible
for the occurrence of OS.
A study published by Huang CH and colleagues in
2011, aimed at proteomic analysis of up-regulated
proteins of Helicobacter pylori under oxidative stress
led to very interesting results [33]. The development ofgastric cancer is known to be associated with chronic
inflammation that occurs as a consequence ofHelicobacter pylori infection. In such inflammations,the oxidative stress induced by reactive oxygen
species in vivo can exert bidirectional effects on both
hosts and Helicobacter pylori. In this study, ROSinduced oxidative stress was mimicked in coculture ofgastric epithelial cells with Helicobacter pylori treatedwith hydrogen peroxide (H2O2). To investigate theeffect of H2O2 on the Helicobacter pylori proteome,
two-dimensional polyacrylamide gel electrophoresis
was performed, followed by liquid chromatography
with mass spectrometry and bioinformatics analysis of
the database. The presence of overexpressed
proteins was found: protein A associated withcytotoxin (CagA), vacuolating cytotoxin (VacA),
adhesion protein (AlpA), two antioxidant enzymes alkylhydroperoxide reductase (AhpC) and catalase(KatA), plus a serine protease (HtrA), aconite hydrate
and fumarate reductase. In addition, up-regulation ofvirulence factors and antioxidant proteins in several
strains of Helicobacter pylori isolated from patients
have been observed. Furthermore, the rate of
Helicobacter pylori infection was found to decrease
and proliferation increase after exposure to H2O2. It
has also been observed that gastric epithelial cells canbe protected from oxidative H2O2 damage in thepresence of Helicobacter pylori. In conclusion, thisstudy supports the assumption that reactive oxygen
species containing H2O2 may induce up-regulation of
Helicobacter pylori virulence factors and antioxidant
enzymes and may explain the inflammation that
contributes to the development of gastric cancer due
to Helicobacter pylori infection.
•

6. Electrochemical sensors
They allow the real-time quantification ofoxidative stress in complex biological systems.
Disadvantages: difficulties in calibration andsometimes operation, in complex biological systems,
due to the interference between the free radicals
formed and their instability.

Fiziologia - Physiology • 2020.30.2(100)CONCLUSION
The development of methods capable of in-depth
analysis of oxidative stress, and of correlatingoxidative stress with the bacterial response could
contribute to obtaining a perspective on thedevelopment of new antibacterial strategies.
Acknowledgement: This work was supported by the
grant “Bioeconomic approach of the antimicrobial
agents - use and resistance – BIO-AMR”, PN-III-P11.2-PCCDI-2017-0361.

REFERENCES
1.

Halliwell B, Gutteridge JMC, Radicalii liberi în
biologie și medicină; in 3rd ed. New York: Oxford
University Press, 1999.
2. Birben E, Sahiner UM, Sackesen C, Erzurum S,
Kalayci O, Oxidative Stress and Antioxidant
Defense. World Allergy Organ J. 2012 Jan; 5(1):9-19.
3. Ezraty B, Gennaris A, Barras F, Collet J-F.
Oxidative stress, protein damage and repair inbacteria. Nature Reviews Microbiology, 2017; 15;385-396.
4. Kim S, Park C, Jang H, B Kim, H Bae, I Chung, E
Kim, Y Cho. Antibacterial strategies inspired by
the oxidative stress and response networks,
Journal of Microbiology, 2019; 57(3): 203-212.
5. Cuzzocrea S, Riley DP, Caputi AP, Salvemini D.
Antioxidant therapy: A new pharmacologicalapproach in shock, inflammation, andischemia/reperfusion injury. Pharmacol Rev,
2001; 53; 135-159.
6. Katerji M, M Filippova, P Duerksen-Hughes.
Approaches and Methods to Measure Oxidative
Stress in Clinical Samples: Research
Applications in the Cancer Field. Oxidative
Medicine and Cellular Longevity, 2019.
7. Seki M, Iida K, Saito M, Nakayama H, Yoshida S.
Hydrogen peroxide production in Streptococcus
pyogenes: involvement of lactase oxidase andcoupling with aerobic utilization of lactate, J.
Bacteriol., 2004; 186; 2046-2051.
8. Winterbourn CC, Hampton MB, Livesey JH,
Kettle AJ. Modeling the reactions of superoxideand myeloperoxidase in the neutrophil
phagosome, Implications for microbial killing, J.
Biol. Chem., 2006; 281; 39860-39869.
9. Imlay JA, Chin SM, Linn S. Toxic DNA damage
by hydrogen peroxide through the Fenton
reaction in vivo and in vitro. Science, 1988; 240;
640-642.
10. Rai P, Cole TD, Wemmer DE, Linn S.
Localization of Fe(2+) at an RTGR sequence
within a DNA duplex explains preferential

65

11.
12.
13.

14.
15.
16.
17.

18.

19.

20.
21.
22.

66

cleavage by Fe(2+) and H2O2, J Mol Biol., 2001;
312; 1089-1101.
Imlay JA. Transcription factors that defend
bacteria against reactive oxygen species. Annu
Rev Microbiol., 2015; 69; 93-108.
Olinescu R. Radicalii liberi în fiziopatologiaumană, Ed. Tehnică, Bucureşti, 1994.
Wei JP, Srinivasan C, Han H, Valentine JS, Gralla
EB. Evidence for a novel role of copper-zincsuperoxide dismutase in zinc metabolism. J Biol
Chem, 2001.
Olanow CW. A radical hypothesis forneurodegeneration. TINS, 1993; 16(11): 439-444.
Jones P. Roles of water in heme peroxidase andcatalase mechanisms. J Biol Chem, 2001;
276(17); 13791-13796.
Lee GY, Han S. The Role of Vitamin E in
Immunity. Nutrients, 2018; 10(11); 1614-1628.
Yang CS, Luo P, Zeng Z, Wang H, Malafa M, Suh
N. Vitamin E and cancer prevention: Studies withdifferent forms of tocopherols and tocotrienols.
Mol Carcinog., 2020; 59(4); 365-389.
El Halabi I, Bejjany R, Nasr R, Mukherji D,
Temraz S, Nassar FJ, El Darsa H, Shamseddine
A. Ascorbic Acid in Colon Cancer: From the Basic
to the Clinical Applications. Int J Mol Sci., 2018;19(9); 2752-2761.
Sies H, Jones DP. Reactive oxygen species(ROS) as pleiotropic physiological signalling
agents. Nat Rev Mol Cell Biol., 2020; 21(7); 363383.
Dahl JU, Gray MJ, Jakob U. Protein quality
control under oxidative stress conditions. J. Mol.
Biol., 2015; 427; 1549-1563.
Palmer LD, Skaar EP. Transition metals andvirulence in bacteria. Annu. Rev. Genet, 2016;
50; 67-91.
Sigler K, Stadler N, Brozmanova J. Oxidative
stress in microoganisms. Folia Microbiologica,
1999; 44(6); 587-624.

23. Marrakchi M, Liu X, Andreescu S. Oxidative
stress and antibiotic resistance in bacterial
pathogens: state of the art, methodologies, andfuture trends. Adv Exp Med Biol., 2014; 806; 48398.
24. Wright GD. On the road to bacterial cell death.
Cell, 2007; 130(5); 781-783.
25. Kohanski MA, Dwyer DJ, Collins JJ. How
antibiotics kill bacteria: from targets to networks.
Nat Rev Microbiol, 2010; 8(6); 423-435.
26. Kohanski MA, et al. A common mechanism ofcellular death induced by bactericidal antibiotics.
Cell, 2007; 130(5); 797-810.
27. Imlay JA. The molecular mechanisms andphysiological consequences of oxidative stress:
lessons from a model bacterium. Nat Rev
Microbiol, 2013; 11(7); 443-454.
28. Yamazaki T, Kawai C, Yamauchi A, Kuribayashi
F. A highly sensitive chemiluminescence assay
for superoxide detection and chronic
granulomatous disease diagnosis. Trop Med
Health, 2011; 39(2); 41-45.
29. Yeom J, Imlay JA, Park W. Iron homeostasis
affects antibiotic-mediated cell death inpseudomonas species. J Biol Chem, 2010;
285(29); 22689-22695.
30. Albesa I, et al. Oxidative stress involved in the
antibacterial action of different antibiotics.
Biochem Biophys Res Commun, 2004; 317(2);605-609.
31. Manoil D, Bouillaguet S. Oxidative Stress in
Bacteria Measured by Flow Cytometry. Adv
Biotech & Micro, 2018; 8(1); 555726.
32. Goswami M, Mangoli SH, Jawali N. Involvementof reactive oxygen species in the action of
ciprofloxacin against Escherichia coli. Antimicrob
Agents Chemother, 2006; 50(3); 949-954.
33. Huang C-H, Chiou S-H. Proteomic analysis of
upregulated proteins in Helicobacter pylori underoxidative stress induced by hydrogen peroxide,
Kaohsiung J Med Sci, 2011; 27(12); 544-553.

Fiziologia - Physiology • 2020.30.2(100)UTILIZAREA DE ASSAY-URI SPECIFICE PENTRU
CUANTIFICAREA STRESULUI OXIDATIV LABACTERIILE MULTIREZISTENTEREZUMATÎn cursul stresului oxidativ echilibrul dintre producția de specii reactive ale oxigenului și sistemele protectoareantioxidante este tulburat. Dintre speciile reactive ale oxigenului (specii chimice care posedă un electron impar)
fac parte: radicalul hidroxil (OH˙), anionul superoxid (O2˙‾), peroxidul de hidrogen (H2O2), în timp ce sistemele
protectoare antioxidante se clasifică în: antioxidanţi enzimatici (superoxiddismutaza, glutation peroxidaza,
glutation S-transferaza) și antioxidanţii neenzimatici (glutationul redus, vitamina E, acidul ascorbic, seleniul, betacarotenii şi vitamina A). În fața invaziei microbiene, celulele se apără eliberând specii reactive ale oxigenului, daraproape toate bacteriile au evoluat și sunt capabile să formeze și să utilizeze o serie de antioxidanți, pentru a
gestiona efectele toxice ale stresului oxidativ. Pentru a înțelege mecanismele de apărare ale bacteriilor s-au utilizat
modele bacteriene care au reușit să caracterizeze răspunsurile adaptative ale acestora. Dezvoltarea metodelorcapabile să coreleze stresul oxidativ cu răspunsul bacterian ar putea contribui la obținerea unei perspective asupra
dezvoltării de noi strategii antibacteriene.
Cuvinte cheie: stres oxidativ, specii reactive ale oxigenului, antioxidanți enzimatici, antioxidanți neenzimatici

Fiziologia - Physiology • 2020.30.2(100)67

IMMUNOCAP ISAC MULTIPLEX MICROARRAY IGETESTING – A TOOL FOR COMPONENT-RESOLVEDDIAGNOSIS IN ALLERGYZBIRCEA LAURIANA-EUNICE1,2,*, GRIJINCU MANUELA1,2,*, BUZANMARIA-ROXANA1,2, GASTAGER FELIX3, CHEN KUAN-WEI1, PANAITESCUCARMEN1,2
1OncoGen-Centre for Gene and Cellular Therapies in the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency County Hospital,

156 Liviu Rebreanu Bv, 300723 Timisoara, RO
2"Victor Babes" University of Medicine and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2, 300041 Timișoara, RO
3HVD Vertriebs-Gesellschaft m.b.H, Vienna, Austria, AT

ABSTRACTIntroduction: Allergy has become a major health concern worldwide, affecting more than 20% of the populationin the industrialized world. Allergy symptoms negatively impact quality of life of the sensitized individuals, increase
in severity over time and with multiple allergen sensitizations. Component-resolved diagnosis can provide insights
into patients’ sensitization profiles, identifying primary sensitizers, cross-reactive components and risk to develop
more severe symptoms in the future.
Aim: To identify IgE repertoires of polysensitized patients and to test whether multiplex ImmunoCAP ISAC assay
can help to differentiate between genuine sensitization and cross-reactivity.
Methods: Three ragweed-allergic patients presenting sensitizations to at least two other allergen sources havebeen included in the study. Serum from these individuals was tested in ImmunoCAP ISAC containing 112 allergencomponents derived from 50 allergen sources according to manufacturer’s instructions.
Results: All patients showed reactivity to the major allergen from ragweed Amb a 1 and had reactivity towards
species-specific components from timothy grass. The main cross-reactive components towards which two patients
showed low to moderate response were profilins, while one patient also had IgE towards tropomyosins. Highest
IgE levels towards these cross-reactive components were for those contained in the same source as speciesspecific components.
Conclusions: ISAC multiplex testing helped to identify sensitization to an allergen source by reactivity towards
the species-specific allergens, while also identifying reactivity towards cross-reactive allergens found in multiple
sources. Additional allergens could improve the identification of primary sensitizers. For proper allergy diagnosis
and treatment, patient history and exposure would also need to be evaluated by the allergist to determine theappropriate immunotherapy for the patient.
Keywords: allergy, IgE, polysensitized, cross-reactivity, ISAC multiplex

INTRODUCTION
Atopy is defined as the disposition to develop IgE
antibodies against otherwise harmless external agents,
which by cross-linking result in the release of
inflammatory mediators like histamines or leukotrienes
by the effector cells [1,2]. In the industrialized world,allergic disease occurs in around 20% of the
population and appears to be increasing [3, 4]. The
most common symptoms of allergic diseases arerhinitis and conjunctivitis [5]. Rhino-conjunctivitis
reduces the quality of life and implies increased healthcosts for sensitized individuals. Additionally, rhinoconjunctivitis can develop into more severe forms ofallergic disease, like asthma [6], while rhinitis andReceived 27th of September 2020. Accepted 9th of October 2020. Address for correspondence: Zbircea Lauriana-Eunice, PhD
student, OncoGen-Centre for Gene and Cellular Therapies in the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency
County Hospital Timisoara, Romania, Liviu Rebreanu No.156 Blv., Timisoara, RO-300723, Romania; phone/fax: +40356433282;
e-mail: lauriana_zbarcea@yahoo.com

68

Fiziologia - Physiology • 2020.30.2(100)asthma is more prevalent among patients sensitized
to more than one allergen source [7]. Patients withmultiple sensitizations and morbidities also have more
persistent and more severe asthma symptoms [6].
Most frequent sensitizations are towards house dust
mites (HDM) and pollen sources like timothy grass andshort ragweed, while most severe allergic
manifestation of allergy, anaphylaxis, may be causedby sensitization to allergens in food, insect venoms,
and drugs [3], [8].
Allergen sources contain multiple proteins withsensitizing potential, some of which are considered
species-specific major allergens and used as markers
for sensitization while others are known panallergens(proteins with functional and structural homology
belonging to the same protein family) (Fig.1). Proteins
which are known as panallergens belong to the profilin,
tropomyosin, polcalcin, nonspecific Lipid Transfer
Protein (nsLTP) and Pathogenesis-Related protein(PR) families [9]. Allergy onset is triggered bysensitization towards the major and some minor
allergens, while sensitization towards panallergens
occurs after high exposure to allergen source and issometimes an indicator of disease severity [9, 10].
When IgE antibodies are raised against a specificantigen, but are able to recognize sequences fromother antigens based on their primary or tertiary
structure, cross-reactivity occurs [11, 12]. Allergens
with an amino acid identity above 70% are often
considered to trigger reactions of cross-reactivity,
while allergens with identities below 50% rarely crossreact [13]. Sequence homology is not always aprerequisite for cross-reactivity, since these reactions
were also found between non-related proteins, i.e. soy
Gly m 6 and cow milk Bos d 6 [12]. These reactions
can be clinically relevant when both sources cantrigger symptoms or they can be irrelevant to thepatient [11]. Patients sensitized to certain pollens haveoften reported symptoms after ingestion of certain
foods, known as pollen-food syndrome (PFS) e.g.
birch pollen allergic patients, sensitized to the major
allergen Bet v 1 report symptoms after ingesting apple,
due to the homology between the two PR-10 proteinsBet v 1 and Mal d 1. Birch immunotherapy was also
found to reduce the symptoms towards apple [14].
Similarly, patients with sensitization to plant foodallergen Pru p 3, nsLTP from peach, also had rhinitis
symptoms in response to mugwort nsLTP Art v 3 [15].
Another common PFS is ragweed-melon-banana
which is probably induced by primary sensitization withreactivity towards nsLTP or profilin from ragweed [16].
Due to its predicted dispersal and increased
allergenicity, ragweed has become a major healthproblem in Europe and worldwide [17]. Therefore it isimportant to investigate potential cross-reactive
Fiziologia - Physiology • 2020.30.2(100)allergen sources among ragweed allergic patients todetermine primary sensitization and select theappropriate immunotherapy [18].
Diagnosis of type I allergy requires thedetermination of allergen-specific IgE (sIgE) in the
blood of patients or induction of immediate symptoms
towards allergen in provocation test (nasal, skin, oral
or bronchial provocation test) [19, 20]. Skin prick test
(SPT) using allergen extracts were preferred byphysicians and patients, since they were a fast, costefficient and easy way to diagnose allergy [21].
However, disadvantages include the lack ofstandardization of allergenic components whenobtaining allergen extracts as well as the presence ofcross-reactive carbohydrate determinants andpanallergens in natural extract which could give false
positive results [22]. To identify the specificsensitization profile of patients, production ofrecombinant allergens has proven to be an invaluable
tool in the component resolved diagnosis of allergy
and precision medicine [19, 23]. Two different
techniques are most frequently employed incomponent-resolved
diagnosis:
singleplex
ImmunoCAP and multiplex ImmunoCAP Immuno
Solid-phase Allergen Chip (ISAC). An important
advantage of ImmunoCAP testing is obtaining
quantified allergen-sIgE levels against an allergencoated on a cellulose matrix without interference from
IgG. On the other hand, ISAC provides an overview of
IgE sensitization patterns towards 112 allergens from50 allergen sources coated on polymer-coated glass
slide using only small quantities of serum. A drawback
for ISAC being that it gives you only semiquantitative
data at a relatively high cost per assay [24, 25]. In
ImmunoCAP the bound IgE antibody is measured withan enzyme labelled anti-IgE antibody, where thefluorescence intensity is proportional to the amount ofbound IgE. The principle behind the multiplex assay issimilar, in ISAC the allergen-bound IgE is measured
using fluorescently labelled anti-human IgE which isdetected using a laser scanner. Both componentresolved assays can provide valuable information
regarding genuine sensitization by the use of marker(species-specific) allergens or enabling to identify
cross-reactivities by sensitization to panallergens.
This assay provides also the opportunity to identify IgEreactivity profiles at risk of developing sever symptoms,
e.g. anaphylaxis [26]. The ISAC assay has recently
been evaluated by different operators in 23 sites
worldwide, showing high repeatability across sites andvariability estimated at 25.5% [27].
Aim of this study is to illustrate the effectivenessof component-resolved diagnosis in deciphering
primary sensitization in ragweed allergic patients
presenting multiple sensitizations in SPT. The patients69

chosen for this assay present multiple sensitizations
towards at least three types of pollen. Therefore, theaim was to test whether ISAC multiplex assay can help
to differentiate between genuine sensitization andcross-reactivity in patients presenting multiple
sensitizations.

allergen source (e.g. ragweed)

manufacturer and three sera from polysensitizedpatients. IgE sensitisation to 112 allergen components
was determined in triplicate by a semi-quantitative
allergen microarray assay using the ImmunoCAP
ISAC 112i (ThermoFisher, Uppsala, Sweden) platform
following the manufacturer’s guidelines (Fig. 2).
CTR03 is composed of known amounts of humanizedchimeric monoclonal antibodies against 16 molecularcomponents (Amb a 1, Art v 1, Art v 3, Bet v 1, Can f
1, Can f 2, Can f 5, Der f 1, Der p 1, Fel d 1, Gal d 1,
Gal d 2, Mal d 1, Pla a 3, Phl p 5, Pru p 1, Pru p 3).
The antibodies against these allergenic components
are found in the calibrator in a range of 4 different
concentrations expressed in ISAC Standardized Units(ISU-E) values for sIgE (1, 4, 15 and 50). The
calibration curve is plotted from the 4 points offluorescence intensity which correspond to thedifferent ISU (Fig. 3 A, B).

allergen components

species-specific components(e.g. Amb a 1)

cross-reactive components(e.g. Amb a 8)

Fig. 1. Component-resolved diagnosis. Allergensources contain multiple allergenic components,
which can be either species-specific or cross-reactive.
MATERIALS AND METHODSPatients sera
Three ragweed-allergic patients were selected forinclusion in this study after examination by an allergist
at the Allergology Unit in Clinics, Timisoara, Romania.
Inclusion criteria were reactivity towards ragweed
pollen extract (SPT or blood IgE) as well as reactivity
towards at least two other allergen sources (pollen
from trees, grasses, other weeds, house dust mites,
fungi, furry animals, latex or food) and symptoms ofallergic rhinitis and conjunctivitis during allergy season.
The participants in this study signed an informed
consent to donate blood voluntarily. Sera samples
were stored at -80°C until use. The study wasapproved by the local ethics committee of County
Emergency Clinical Hospital „Pius Brînzeu” Timisoara(Ethical approval number 102, 10.01.2017).

Fig. 2. ImmunoCAP ISAC array layout. Layout shows
positions and names of 112 allergen components(MIA software)

Determination of IgE profiles using ISAC 112i
For this study four samples were measured: thecalibrator sample (CTR03), provided by the
70

Fiziologia - Physiology • 2020.30.2(100)The microarray was incubated with 30 µL ofpatient serum for 120 minutes at room temperature.
Slides were then washed and 30 µL fluorescencelabelled human IgE antibody was added on eachmicroarray. After 30 min incubation in the darkhumidity chamber, unbound antibodies were removed
by washing and the fluorescence intensity wasmeasured by microarray scanner (LuxScan 10K/A,
CapitalBio, Beijing, China) with the following
parameters: laser power (LP = 60) and photo-multipliertube (PMT = 600). Results were interpreted using
Phadia Microarray Image Analyzer software (MIA,
ThermoFisher). The sIgE values expressedsemiquantitatively as ISU-E units are assigned to oneof the following four categories: Undetectable (<0.3
ISU-E), Low (0.3-0.9 ISU-E), Moderate/High (1-14.9
ISU-E), Very high (≥15 ISU-E). Phadia Xplain add-on(ThermoFisher Uppsala, Sweden) was used for datainterpretation.
Fig. 3A. ImmunoCAP ISAC image scan of thecalibrator sample (CTR03). Allergen components arearrayed in vertical triplicates. Image is shown in false
colour display mode to better visualize thefluorescence output. Linear scale of false colour
display is shown below the image.

RESULTS
All patients had a positive response in skin prick
test/ blood IgE test towards weed pollen and grasspollen, while patients 2 and 3 were also reactive
towards tree pollen (Table I.). Patient 1 also had
positive results towards house dust mites (HDM), fungi,
furry animals and food, while patient 2 was positive
towards latex extract and HDM (Table I).

Table I. Patients characterization and sensitization todifferent allergen sources. Patients sensitization
towards extracts as evaluated at the allergist clinic. (+positive, - negative, NA not available)

P
a
t.

1

.

Fig. 3B. ISAC calibration curve used for thisassay analysis (CTR03)

Fiziologia - Physiology • 2020.30.2(100)

2
3

A S
g e
e x

T
r
e
e

3
9 M 6
3 M +
1
6 F +

G
ra
ss

Allergen sources
Furr
W Hous
F
y
e
e
u
ani
e
Dust
n
mal
d
Mite
gi
s

+

+

+

+

+

+

+

+

-

+

+

NA

N
A

NA

L
at
e
x

F
o
o
d

N
A

+

+
N
A

N
A

71

Component resolved analysis revealed that all
three ragweed-allergic patients (patient 1, 2 and 3)
were positive (ISU-E≥0.3 ISU-E) for major ragweed
component Amb a 1 with measured values between
8.8-20 ISU-E, corresponding to moderate – very highresponse (Fig. 4 A-F). Patient 2 reacted to major
component from mugwort Art v 1 with a value of 3 ISUE (Fig. 4 D). Patients 1 and 2 also reacted with profilin
Mer a 1 from annual mercury with values of 0.5 and8.7 ISU-E respectively (Fig. 4 B, D).
All three patients with allergy to grass pollen
reacted to one or more species-specific components
from timothy grass pollen as follows: patients 1 and 3
reacted to Phl p 1 with values of 2.3 and 71 ISU-E, to
Phl p 2 with values of 0.9 and 17 ISU-E, patients 2 and3 reacted to Phl p 4 with values of 1.6 and 2.3 ISU-E,
patient 3 reacted to Phl p 5 and Phl p 6 with values of
25 and 5.2 ISU-E. Patient 2 also reacted to crossreactive profilin Phl p 12 with a value of 1.7 ISU-E (Fig.
4 D). Cyn d 1 resulted in a moderate IgE response inpatient 2 (2 ISU-E) and a very high response in patient
3 (36 ISU-E) (Fig. 4 C-F).
Among the patients sensitised to tree pollen,patient 3 showed reactivity to major allergencomponent from birch Bet v 1 with a value of 1.1 ISUE, whereas patients 1 and 2 showed reactivity to
profilin Bet v 2 with values of 0.4 and 7.6 ISU-E (Fig. 4
A-F). Patient 3 reacted slightly to major component
from Japanese cedar Cry j 1 with value of 0.9 ISU-E.
Patient 2 showed a positive response to allergens
from house dust mites (HDM) and mould (Fig. 4 D).
Species-specific allergens Der f 2 and Der p 2 resulted
in moderate response with values of 3,8 and 4,6 ISUE, while the cross-reactive component tropomyosin,
Der p 10, also had a moderate value of 1.7 ISU-E.
Patient 2 also reacted to tropomyosins from threeother animal sources: anisakis Ani s 3, cockroach Bla
g 7 and shrimp Pen m 1 with low to moderate valuesof 0.8, 0.9 and 1.2, respectively (Fig. 4 D). Patient
showed low reactivity to the enolase from mould Alt a
6 with a value of 0.6 ISU-E.
Other reactivities in ISAC were to profilin fromlatex Hev b 8 for patients 1 and 2 with values of 0.4
and 8.4 ISU-E and to food allergen from hazelnut Cor
a 1.0401 for patient 3 with a value of 0.4 ISU-E. Also,patient 2 and 3 showed reactivity to CCD (crossreactive carbohydrate determinant) MUXF3 with lowlevels of 0.7 and 0.9, respectively (Fig. 4 D, F).

72

Fig. 4. ISAC IgE measurements for the three patients.
ISAC readings and average ISAC Standardized Units(ISU-E) results for patient 1 (A, B), patient 2 (C, D) andpatient 3 (E, F).

DISCUSSION
ImmunoCAP ISAC Molecular Allergy Test has
proven to be a highly sensitive and specific method of
diagnosis in allergy, showing a strong correlation withother methods as SPT and sIgE tests [27, 28]. Studies
found that in Europe individuals are exposed tooverlapping pollen seasons of a variety of allergenic
plants [29]. Ragweed, mugwort, birch and timothy
grass are only a few of the most common pollen
sources which can be found in Western Romania [30,31]. Previous studies conducted in Timisoara,
Romania, showed that ragweed allergy has the
Fiziologia - Physiology • 2020.30.2(100)highest prevalence in both monosensitized and inpolysensitized patients and ragweed pollen isconsidered to be the most significant outdoor allergen[32]. Therefore, three patients with ragweed allergy inaddition to at least two other sensitizations werechosen to investigate allergen-specific IgE repertoire
to 112 components from 50 allergen sources.
The sensitisation rate for the three patients
included in our study was higher for pollen-derived
components than for non-pollen derived components.
All three ragweed-allergic patients showed reactivity to
the species-specific component Amb a 1 fromragweed with high IgE levels, as expected, since older
studies show that Amb a 1 is a highly allergenic
molecule recognised by more than 90% of the
ragweed-allergic patients and high IgE titres are foundin patient sera [33]. Although other major and minor
allergens are known from ragweed pollen to beclinically relevant contributing to the clinical picture of
ragweed allergy, in this case, the patients are
genuinely sensitised to ragweed pollen. In addition, allpatients also reacted to at least one component fromtimothy grass.
Patient 1 showed specific IgE to major allergens
Phl p 1 and Phl p 2 which indicate genuine
sensitisation to timothy grass [34]. Low IgE reactivity
to the profilins from annual mercury Mer a 1, latex Hev
b 8 and birch Bet v 2, can indicate cross-reactivity due
to the high sequence homology of these proteins.
Studies found that Bet v 2 was an indicator of crossreactivity with profilins from other pollen sources,
plant-based foods and latex, similar to Mer a 1 whichcross-reacted with other pollen profilins and Hev b 8
which was found to cross-react with other pollen
profilins rather than to indicate genuine latex
sensitization [34, 35]. All this data suggests that thispatient may be sensitised to other profilins which are
currently not spotted on the chip. The reactivity to thebeforementioned profilins and not Phl p 12 (fromtimothy grass) could be attributed to sensitization
towards profilin Amb a 8, a minor allergen from theragweed pollen with an IgE frequency of 20-35 % inragweed-allergic patients and a sequence identity ofover 65% with other known allergenic profilins [36].
However, profilin sensitization was indicated to havelittle clinical relevance in allergic diseases [37].
Patient 2 appeared to be sensitised to Bermuda
grass and timothy grass due to the positive response
to natural components Cyn d 1 and Phl 4, respectively.
This patient also had low IgE level to CCD. Proteins
purified from extract can contain CCD components
and IgE reactivity to Bermuda grass Cyn d 1, timothy
grass Phl p 4, cypress Cup a 1, Japanese cedar Cry j
1 and plane Pla a 2 could be partially based on crossreactivity to these CCDs [38, 39]. Although more thanFiziologia - Physiology • 2020.30.2(100)20% of the allergic patients were found to have IgEthat binds to CCDs, the clinical relevance of thesemolecules has yet to be proven [39]. Both Cyn d 1 andPhl p 4 are considered species-specific allergens andin this case, patient 2 is probably sensitised to both
sources with only a small part of the IgE to Cyn d 1
and Phl p 4 being attributed to CCDs [34]. Art v 1 is themain specific mugwort component as at least 95% of
the mugwort allergic patients react to this allergen and
IgE to this marker shows sensitisation to mugwort
pollen [34]. However, in ragweed pollen (towards
which the patient is sensitized according to the highreactivity to Amb a 1) there is a minor allergen(defensin Amb a 4) 1 which is not included on the chip,showing 50% sequence identity with Art v 1 [36].
Pablos et al. found that between 52 and 74% of
patients which reacted to Art v 1 (depending on region)
also responded with a similar intensity towards thehomologs in ragweed and Parthenium [40].
Patient 2 showed low reactivity to Alternaria
enolase Alt a 6 minor allergen indicating probably
weak sensitisation to mould [41]. Specific IgE towards
marker allergens Der f 2 and Der p 2 indicated genuine
sensitisation to house dust mites (HDM). Der p 2 andDer f 2 can cross-react. This patient also showed lowmoderate reactivity to tropomyosins Ani s 3, Bla g 7
and Pen m 1, which could be due to cross-reactivity to
Der p 10 from house dust mites towards which thepatients is sensitized and which had slightly higher IgElevel [34]. In order to determine the primary sensitizer
in case of cross-reactivity, the highest IgE values for a
protein from that family has been suggested asindicator of primary sensitization, while lower IgEvalues may be to cross-reactive proteins [38].
This patient also showed IgE reactivity to crossreactive components from birch Bet v 2, latex Hev b 8,
annual mercury Mer a 1 and timothy grass Phl p 12
indicating cross-reactivity between these profilins [34].
Based on the IgE levels towards these components, it
is likely that patient 2 was primarily sensitized toannual mercury and this led to cross-reactivity withother profilins. On the other hand, given that patient 2
appeared to be sensitized to timothy grass, Phl p 12
could also be considered as primary sensitizer,
although this could not explain why the IgE value to
Phl p 12 was four or five times lower than to Bet v 2,
Hev b 8 and Mer a 1. Another possibility could be thatpatient 2 was primarily sensitized to profilin from otherallergen sources known to cross-react and towards
which that patient showed high IgE reactivity, butwhich are unfortunately not available on the chip for
testing (e.g. ragweed Amb a 8 or mugwort Art v 4).
Patient 3 showed a high reactivity towards the
species-specific components from grass pollen, fivemainly species-specific components of timothy grass73

(Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6) indicating
genuine sensitization to timothy grass [42, 43]. HighIgE values to Bermuda grass Cyn d 1 could also
indicate a genuine sensitization to Bermuda grass,since Cyn d 1 is considered representative marker
allergen for this species [34]. However, we also haveto consider that this reactivity may be due to crossreactivity to timothy grass Phl p 1. Previous studies
have found a high degree of allergenic similarity or
cross-reactivity between Bermuda grass pollen andtimothy grass pollen and IgE to Cyn d 1 may crossreact to Phl p 1 [38, 44, 45].
Patient 3 showed a weak sensitization to tree pollen.
IgE reactivity to Bet v 1 component indicatessensitization to birch [46]. IgE reactivity to hazelnut
Cor a 1.0401 is associated with local allergic reactions
in patients with pollen-food-syndrome caused bysensitization to birch (e.g. Bet v 1) or related trees [34].
In this case, sensitization to Cor a 1.0401 could be due
to cross-reactivity to birch Bet v 1 belonging to thesame protein family (PR-10 protein). Part of the IgEreactivity to components Cyn d 1 and Phl p 4 for thispatient could be due to IgE against CCD, asmentioned before. Since the IgE values to Japanesecedar Cry j 1 and CCD are identical, it is possible thatpatient 3 was not primarily sensitized to Cry j 1 and thepositive response could be entirely due to the CCDspecific IgE [34].

Prevention, diagnosis and therapy of ragweed pollen
Induced REspiratory Diseases) project, COP 20142020 92/09.09.2016, P_37_747.

REFERENCES
[1]
[2]

[3]

[4]
[5]

[6]

[7]

CONCLUSIONISAC multiplex assay has proven to be an
important tool to determine the IgE profiles of thepolysensitized patients included in our study. Afterdata interpretation, patients included in this study weresensitized to fewer allergen sources according to chipthan was previously determined by extract testing.
Therefore, ISAC measurements could help to explain
certain reactivities obtained when testing the allergen
extracts due to the cross-reactive allergencomponents or carbohydrate determinants withinthose allergen sources. Additional allergens on thechip could provide more information regarding primarysensitizer among cross-reactive allergens, especiallywhen considering the exposure of the patients to theallergens. This information is very useful for precision
medicine and personalized treatment in allergy.
However, for the clinical diagnosis and selection ofallergen immunotherapy, patient and symptom history
as well as allergen exposure will have to be evaluatedby the allergist to establish the appropriate treatment
for each patient.
ACKNOWLEDGEMENTS: This research was
financed by the INSPIRED (INovative Strategies for
74

[8]

[9]

[10]

[11]

[12]
[13]
[14]

B. A. Kay, A. P. Kaplan, J. Bousquet, and P. G. Holt,
Allergy and Allergic Diseases 2nd Edition, vol. 1.
2009.
D. M. Segal, J. D. Taurog, and H. Metzger,“Dimeric immunoglobulin E serves as a unit signal
for mast cell degranulation,” Proc. Natl. Acad. Sci.,
vol. 74, no. 7, pp. 2993–2997, 1977.
S. J. Arbes Jr., P. J. Gergen, L. Elliott, and D. C.
Zeldin, “Prevalences of positive skin test
responses to 10 common allergens in the US
population: Results from the Third National Healthand Nutrition Examination Survey,” J. Allergy Clin.
Immunol., vol. 116, no. 2, pp. 377–383, Aug. 2005.
R. Valenta et al., “From allergen genes to allergy
vaccines,” Annu. Rev. Immunol., vol. 28, pp. 211–
241, 2009.
D. I. Bernstein, G. Schwartz, and J. A. Bernstein,“Allergic rhinitis: mechanisms and treatment,”
Immunol. Allergy Clin., vol. 36, no. 2, pp. 261–278,
2016.
J. M. Anto et al., “Mechanisms of the developmentof allergy (MeDALL): introducing novel concepts inallergy phenotypes,” J. Allergy Clin. Immunol., vol.
139, no. 2, pp. 388–399, 2017.
V. Siroux et al., “The asthma‐rhinitis multimorbidity
is associated with IgE polysensitization inadolescents and adults,” Allergy, vol. 73, no. 7, pp.
1447–1458, 2018.
L. B. Grabenhenrich et al., “Anaphylaxis in children
and adolescents: the European Anaphylaxis
Registry,” J. Allergy Clin. Immunol., vol. 137, no. 4,
pp. 1128–1137, 2016.
O. E. McKenna, C. Asam, G. R. Araujo, A. Roulias,
L. R. Goulart, and F. Ferreira, “How relevant ispanallergen sensitization in the development ofallergies?,” Pediatr. Allergy Immunol., vol. 27, no.
6, pp. 560–568, 2016.
L. Hatzler et al., “Molecular spreading and predictive
value of preclinical IgE response to Phleum pratense
in children with hay fever,” J. Allergy Clin. Immunol.,vol. 130, no. 4, pp. 894–901, 2012.
F. Ferreira, T. Hawranek, P. Gruber, N. Wopfner,and A. Mari, “Allergic cross-reactivity: From geneto the clinic,” Allergy Eur. J. Allergy Clin. Immunol.,vol. 59, no. 3, pp. 243–267, 2004.
M. Bublin and H. Breiteneder, “Cross‐reactivities
of non‐homologous allergens,” Allergy, 2019.
R. C. Aalberse, “Structural biology of allergens,” J.
Allergy Clin. Immunol., vol. 106, no. 2, pp. 228–
238, 2000.
J. Holm et al., “Molecular basis of allergic crossreactivity between group 1 major allergens frombirch and apple,” J. Chromatogr. B Biomed. Sci.
Appl., vol. 756, no. 1–2, pp. 307–313, 2001.
Fiziologia - Physiology • 2020.30.2(100)

[15]

[16]
[17]
[18]

[19]

[20]
[21]

[22]

[23]

[24]
[25]

[26]

[27]

[28]

[29]
[30]

J. S. Sanchez-Lopez et al., “Role of Art v 3 inpollinosis of patients allergic to Pru p 3,” J. Allergy
Clin. Immunol., vol. 133, no. 4, pp. 1018–1025,
2014.
F.-D. Popescu, “Cross-reactivity between
aeroallergens and food allergens,” World J.
Methodol., vol. 5, no. 2, p. 31, 2017.
I. R. Lake et al., “Climate change and future pollen
allergy in Europe,” Environ. Health Perspect., vol.
125, no. 3, pp. 385–391, 2016.
Y. Dorofeeva et al., “Past, presence, and future ofallergen immunotherapy vaccines,” Allergy Eur. J.
Allergy Clin. Immunol., no. February, pp. 1–19,
2020.
R. Valenta, J. Lidholm, V. Niederberger, B. Hayek,
D. Kraft, and H. Grönlund, “The recombinant
allergen-based concept of component-resolved
diagnostics and immunotherapy (CRD and CRIT),”
Clin. Exp. Allergy, vol. 29, no. 7, pp. 896–904,
1999.
R. Valenta et al., Molecular Aspects of Allergens
and Allergy, 1st ed., vol. 138. Elsevier Inc., 2018.
E. Jensen-Jarolim, A. N. Jensen, and G. W.
Canonica, “Debates in allergy medicine: Molecularallergy diagnosis with ISAC will replace screenings
by skin prick test in the future,” World Allergy
Organ. J., vol. 10, no. 1, pp. 1–6, 2017.
R. Valenta et al., “Allergen Extracts for In Vivo
Diagnosis and Treatment of Allergy: Is There a
Future?,” J. Allergy Clin. Immunol. Pract., vol. 6,
no. 6, pp. 1845-1855.e2, 2018.
M. Curin, V. Garib, and R. Valenta, “Single
recombinant and purified major allergens andpeptides: How they are made and how theychange allergy diagnosis and treatment,” Ann.
Allergy, Asthma Immunol., vol. 119, no. 3, pp. 201–
209, 2017.
R. Hiller et al., “Microarrayed allergen molecules:
diagnostic gatekeepers for allergy treatment,”
FASEB J., vol. 16, no. 3, pp. 414–416, 2002.
M. van Hage, C. Hamsten, and R. Valenta,“ImmunoCAP assays: Pros and cons inallergology,” J. Allergy Clin. Immunol., vol. 140, no.
4, pp. 974–977, 2017.
V. Cardona and I. J. Ansotegui, “Componentresolved diagnosis in anaphylaxis,” Curr. Opin.
Allergy Clin. Immunol., vol. 16, no. 3, pp. 244–249,
2016.
M. van Hage et al., “Performance evaluation of
ImmunoCAP® ISAC 112: a multi-site study,” Clin.
Chem. Lab. Med., vol. 55, no. 4, pp. 571–577,
2017.
P. Panzner, M. Vachová, P. Vítovcová, P. Brodská,
and T. Vlas, “A comprehensive analysis of middleEuropean molecular sensitization profiles to pollen
allergens,” Int. Arch. Allergy Immunol., vol. 164, no.
1, pp. 74–82, 2014.
G. D’Amato et al., “Allergenic pollen and pollen
allergy in Europe,” Allergy, vol. 62, no. 9, pp. 976–
990, 2007.
N. Ianovici, C. B. Panaitescu, and I. Brudiu,

Fiziologia - Physiology • 2020.30.2(100)

[31]

[32]

[33]

[34]
[35]

[36]

[37]

[38]

[39]
[40]

[41]

[42]

[43]

“Analysis of airborne allergenic pollen spectrum for2009 in Timişoara, Romania,” Aerobiologia(Bologna)., vol. 29, no. 1, pp. 95–111, 2013.
N. Ianovici, M. Juhasz, A. Kofol-Seliger, and B.
Sikoparija, “Comparative analysis of some vernal
pollen concentrations in Timisoara (Romania),
Szeged (Hungary), Novi Sad (Serbia) andLjubljana (Slovenia),” Not. Bot. Horti Agrobot. ClujNapoca, vol. 37, no. 2, pp. 49–56, 2009.
C. Panaitescu, N. Ianovici, L. Marusciac, L.
Cernescu, P. Tamas, and R. A. Lazarovicz,“Sensitisation to Ambrosia pollen and otherairborne allergens in the population of the Westernregion of Romania: 1181,” Allergy Eur. J. Allergy
Clin. Immunol., vol. 69, 2014.
R. Asero, N. Wopfner, P. Gruber, G. Gadermaier,and F. Ferreira, “Artemisia and Ambrosia
hypersensitivity:
co‐sensitizationorco‐recognition?,” Clin. Exp. Allergy, vol. 36, no. 5, pp.
658–665, 2006.
J. Kleine-Tebbe and T. Jakob, Molecular allergy
diagnostics: innovation for a better patientmanagement. Springer, 2017.
N. Cabañes, J. M. Igea, B. de La Hoz, P. Agustín,
C. Blanco, and J. Domínguez, “1 Latex Allergy:
Position Paper,” J. Investig. Allergol. Clin.
Immunol., vol. 22, no. 5, p. 313, 2012.
K.-W. Chen, L. Marusciac, P. T. Tamas, R. Valenta,
and C. Panaitescu, “Ragweed Pollen Allergy:
Burden, Characteristics, and Management of an
Imported Allergen Source in Europe,” Int. Arch.
Allergy Immunol., vol. 176, no. 3–4, pp. 163–180,
2018.
M. Wensing et al., “IgE to Bet v 1 and profilin:cross-reactivity patterns and clinical relevance,” J.
Allergy Clin. Immunol., vol. 110, no. 3, pp. 435–442,
2002.
J. Kleine-Tebbe and U. Jappe, “Molecular allergy
diagnostic tests: development and relevance inclinical practice,” Allergol. Sel., vol. 1, no. 2, p. 169,
2017.
F. Altmann, “The role of protein glycosylation inallergy,” Int. Arch. Allergy Immunol., vol. 142, no. 2,
pp. 99–115, 2007.
I. Pablos et al., “Distinct epitope structures ofdefensin‐like proteins linked to proline‐rich regions
give rise to differences in their allergenic activity,”
Allergy, vol. 73, no. 2, pp. 431–441, 2017.
I. Kustrzeba-Wójcicka, E. Siwak, G. Terlecki, A.
Wolańczyk-Mędrala, and W. Mędrala, “Alternaria
alternata and its allergens: a comprehensive
review,” Clin. Rev. Allergy Immunol., vol. 47, no. 3,
pp. 354–365, 2014.
R. E. Rossi, G. Monasterolo, and S. Monasterolo,“Measurement of IgE antibodies against purified
grass‐pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11,
and 12) in sera of patients allergic to grass pollen,”
Allergy, vol. 56, no. 12, pp. 1180–1185, 2001.
A. Mari, “Skin test with a timothy grass (Phleum
pratense) pollen extract vs. IgE to a timothy extract
vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl

75

[44]

[45]

p 6, rPhl p 7, rPhl p 11, and rPhl p 12:
epidemiological and diagnostic data,” Clin. Exp.
Allergy, vol. 33, no. 1, pp. 43–51, 2003.
R. E. Rossi, G. Monasterolo, P. Prina, G. Coco, D.
Operti, and L. Rossi, “IgE profiles of Bermuda
Grass Pollen sensitised patients evaluated by
Phleum pratense allergens Phl p 1, 2, 4, 5, 6, 7, 11,12,” Allergol. Int., vol. 57, no. 2, pp. 157–164, 2008.
F. Cipriani et al., “Diagnostic relevance of IgE[46]

sensitization profiles to eight recombinant Phleum
pratense molecules,” Allergy, vol. 73, no. 3, pp.
673–682, Mar. 2018.
P. Comite et al., “Birch allergy and oral allergy
syndrome: The practical relevance of serum
immunoglobulin E to Bet v 1.,” in Allergy & Asthma
Proceedings, 2016, vol. 37, no. 1.

TESTAREA DE ANTICORPI IGE CU MULTIPLEXIMMUNOCAP ISAC MICROARRAY – O METODĂ DE
DIAGNOSTIC MOLECULAR ÎN ALERGIIREZUMAT
Introducere: Alergiile au devenit o problemă majoră de sănătate în toată lumea, afectând mai mult de 20% din
populația din lumea civilizată. Simptomele alergiei reduc calitatea vieții pacientului și manifestarea lor devine tot
mai severă pe măsura creșterii ratei sensibilizării la alte alergene. Diagnosticul molecular oferă o înțelegere maiamplă a profilului de sensibilizare al pacientului, ajută la identificarea sursei sensibilizării primare, a componenteloralergenice care pot da reactivitate încrucișată și la evaluarea riscului de a dezvolta simptome mai severe pe viitor.
Scop: Studiul de față propune identificarea repertoriului de anticorpi IgE al pacienților polisensibilizați prin utilizarea
tehnicii ImmunoCAP ISAC pentru a face diferența între sensibilizarea primară și reactivitatea încrucișată.
Metode: Trei pacienți alergici la polen de ambrozia care au prezentat și simptome la cel puțin alte două surse
alergenice au fost incluși în acest studiu. Serul de la acești indivizi a fost testat în ImmunoCAP ISAC la 112
componente din 50 de surse alergenice conform instrucțiunilor producătorului.
Rezultate: Toți pacienții au prezentat reactivitate la alergenul major din ambrozia și la anumite alergene dintimoftică. Principalele alergene cu potențial de reactivitate încrucișată la care pacienții au prezentat un răspuns
slab sau moderat au fost profilinele; un singur pacient a prezentat reactivitate la tropomiozine. În cazul pacienților
care au răspuns la mai multe alergene omolage, răspunsul IgE cel mai crescut a fost pentru sursa la care pacientul
s-a sensibilizat inițial la alergene majore.
Concluzii: Testarea cu ajutorul tehnologiei ISAC a ajutat la identificarea sursei de sensibilizare prin determinarea
reactivității față de alergenele specifice unei surse, dar și la identificarea alergenelor care dau reactivitateîncrucișată. Adăugarea suplimentară pe cip a altor alergene minore ar putea contribui la identificarea sursei
alergenice primare în cazul reacțiilor de reactivitate încrucișată. În vederea stabilirii unui diagnostic corect alalergiei, istoricul pacientului și expunerea la diferite surse alergenice trebuie evaluată de medicul alergolog în cazul
administrării imunoterapiei adecvate pentru fiecare pacient.
Cuvinte cheie: alergie, IgE, polisensibilizare, reactivitate încrucișată, ISAC multiplex

76

Fiziologia - Physiology • 2020.30.2(100)POST TONSILLECTOMYCOHORT STUDY

PAIN

IN

ADULTS–A

TRANDAFIR CORNELIA-MARINA1,2, MARIN KARINA CRISTINA1,2,
LUPESCU STELIAN1,2, JUCHIAC ANITA VARVARA1, MARIN HORIA
ALIN1,2“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
Clinical Municipal Emergency Hospital Timisoara – ENT Clinics

ABSTRACT
Tonsillectomy is one of the most common procedures performed annually. Pain is reported in all patients in the
postoperative period and persists for a various extent of time. Numerous patients address themselves to theemergency department after a tonsillectomy for pain relief. The aim of the study is to compare the effect ofIbuprofen vs Paracetamol vs Tramadol in the post-operative recovery after tonsillectomy and their link topostoperative bleeding.
Keywords: tonsillectomy, AINS, acetaminophen, tramadol, bleeding

INTRODUCTION
Tonsillectomy is one of the most frequently
performed surgical procedure in the ENT specialty. In
the United States, more than half a million of thepediatric population undergo this surgical procedureeach year [1]. This procedure is usually performed incase of recurrent tonsillitis or complications of tonsillitis(peritonsillar abscesses or deep neck infections), forthe treatment of OSAS or in case of tonsillar
malignancy [2,3].
One of the most postoperative complaints of thepatients after the intervention is pain. The intensity ofpain depends on the surgical technique and the
medical postoperative pain relief treatment. The pain
after a surgical procedure is due to the surgicaldamage to the mucosa and the irritation of the nerve
fibers in the peritonsillar space [4]. Various analgesic
protocols have been used to reduce the post
tonsillectomy pain but significant gaps remain in theliterature as a result of the lack of evidence-based
criteria for the ideal treatment of pain and the link
between some antalgic treatments and postoperatory

bleeding.
Acetaminophen (paracetamol) is commonly used
for surgical pain as well as for analgesic andantipyretic properties [5]. The potential for hepatic
injury exists and has been found in some young
patients [6].
Ibuprofen is a NSAID well known for his great
analgesic effect but also for the increased risk of
bleeding. Ibuprofen acts by inhibiting thecyclooxygenase enzymes resulting in a thromboxane
inhibition (an important component of the coagulation
process) [7].
Opioids are widely used in postoperative, treating
the acute pain. However, they are well known for their
many side effects such as vomiting, nausea, sedation[8]. The use of opioids in the pediatric population isunder discussion and research because they can
produce severe respiratory distress and in some cases
death by the rapid conversion of codeine to morphine
via the CYP2D6 pathway [9].
The aim of our study was to compare the efficacyof the Acetaminophen vs Ibuprofen vs Tramadol forthe treatment of post-tonsillectomy pain in adults andthe incidence of complications such as post-

Received 5th of October 2020. Accepted 11th of November 2020. Address for correspondence: Trandafir Cornelia, “Victor
Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, Romania; Clinical
Municipal Emergency Hospital Timisoara – ENT Clinics, Hector No.1 Street, Timisoara, Romania, phone/fax: +40256 221553 /+40256 495339; e-mail: trandafir.cornelia@gmail.com

Fiziologia - Physiology • 2020.30.2(100)77tonsillectomy bleeding.

MATERIAL AND METHODS
Three groups were compared regarding the pain
severity and the post-tonsillectomy occurrence
hemorrhage. The groups were sorted according togender and age with a similar distribution in thecohorts. The aims were defined as readmission for
pain control, readmission for post tonsillectomy
bleeding and surgical hemostasis.
The indication for tonsillectomy was repetitive
tonsillitis. The cold dissection technique of all thetonsillectomies was performed by the same surgeon
for the entire duration of the study. The hemostasis
was realized by compression and bipolar cauterization.
The postoperative control was made by the sameteam of surgeons and resident doctors. None of thepatients received preoperative or intraoperative
antibiotics.
The patients were appointed every 3 days for a
medical visit and they filled a questionnaire for theevaluation of the pain (a scale from 1 to 10). No patient
missed their appointment. The questionnaire filled by
the patients included also items about the difficulty ofswallowing (for liquids, for solids, for both), andexperience of nausea and other possible side-effectsof the treatment.
The patients with a history of coagulopathy, renal
or liver diseases, peptic ulcer disease, and intolerance
to NSAIDs, asthma or allergies to the substances usedin this research had been excluded from this study.
The patient with acute infections had been also
rejected from the inclusion of this study.
To every age cohort group was appointed asimilar number of patients treated with a different pain
relief drug (Table I).
Age
31-40
years old
41-50
years old
51-60
years old
Total

Acetami
nofen
2

Ibupr
ofen
2

Tram
adol
2

Tot
al
6

2

2

2

6

2

2

2

6

6

6

6

18

Our standard treatment for post tonsillectomy
analgesia has been: Acetaminofen 1 g every 6 h for a
maximum of 4 grams per day, Ibuprofen 400 mg every
12 h for a maximum of 800 mg per day, and Tramadol
5 mg every 6 h for a maximum of 200 mg per day. All
the treatments were prescribed for 12 days. The
monitoring of the patients was over a period of 21 days,

78

with a medical appointment every 3 days.
Our goals were to assess the surgical hemostasis
readmission because of a post tonsillectomy bleeding
and readmission for pain control. In our opinion, thesurgical hemostasis is a more reliable indicator of
bleeding events because some patients might report a
bleeding without the evidence by the surgeon. The
medical team remained the same through the durationof the study in order to have the same objective
criteria’s.
The incidence of the tonsillectomy hemorrhage
was 16% in the Tramadol group vs 0% in the Ibuprofen
group and 33.3% in the Acetaminophen group. The
incidence of re-intervention for post-tonsillectomy
bleeding was 16.67%.
Some studies indicated that the risk of post
tonsillectomy bleeding is increasing with age. Our
study is in agreement with observation because 2 outof the 6 patients with an age between 51-60 years old
were readmitted in the hospital during the 7-10
postoperative days for reintervention. Both of themwere under Acetaminophen treatment [10].
One of the 6 patients treated with Tramadol
presented with postooperatory bleeding, he wasincluded in the 41-50 years age group.
One patient treated with Ibuprofen was
readmitted for post tonsillectomy pain despite theibuprofen treatment. He was in the 31-40 years cohort
group of patients.
Pain intensified in our patients during the 5th to 7thday. In this period, one of the patients (under Ibuprofen
treatment) was readmitted because he presented
dehydration as a consequence of the intensification of
the pain (10 /10 in the questionnaire at day 6). The
Tramadol group of patients had the best pain control(the highest grade was 5/10). The Acetaminophen
proved to reduce pain after tonsillectomy (the highest
grade was 7/10).

DISCUSSION
Post tonsillectomy pain is a significant morbidity
among patients undergoing this intervention making
the pain control and the risk of bleeding the mostcommon concern among surgeons. Poor analgesia
may cause dysphagia, dehydration and leads patients
to emergency medical visits and readmissions [11].
Acetaminofen is widely used after tonsillectomy
due to their analgesic effect comparable with theopioid but without the gastro-intestinal side-effects [12].
The Ibuprofen, a NSAID well known medication,
has the ability to reduce pain and inflammation without
the side effects of the opioid treatment. Yet, the use of
ibuprofen after tonsillectomy remains controversial
due to the increased bleeding time. A study by Krishna
Fiziologia - Physiology • 2020.30.2(100)et al. shows that the bleeding time in a patient withnormal coagulation pathway remains normal under theuse of the NSAID medication. This study is supported
by many trials in the existing literature that assessedthat the use of Ibuprofen is not influencing the post
tonsillectomy bleeding [13,14]. Our study supports thistheory. Only one of the 6 patients treated with
Ibuprofen was readmitted in the hospital for the pain
control (and not for a bleeding as we expected).
Opioid pain control has been used for the
management of pain with reports of many side-effects
and also fatalities in children as a result of geneticallyaltered metabolism of codeine-morphine in thepediatric population [15].
Our research compared by a randomized trial theefficacy of Paracetamol vs Ibuprofen vs Tramadol forthe treatment of post tonsillectomy pain in adults overa 21 days period. Our results indicated that the best
pain relief was achieved by Tramadol. However, all
the patients included in our study described the pain
after tonsillectomy to be less intense than the pain theyfelt each time during the acute tonsillitis.
Our study has several limitations. It is based on asmall population number and thus it lacks of statisticalpower. Further, a larger study has to be made.

4.

5.

6.
7.

8.

9.
10.

11.

CONCLUSION
The pain management of post tonsillectomy pain
is challenging and needs to find a balance between theefficacy controlling of the pain and the possible side
effects. Further larger studies are required to findthe best adequate treatment with the less side effects.

12.

13.

REFERENCES
1.

2.

3.

Baugh R, Archer S, Mitchell R, et al. Clinicalpractice guideline: tonsillectomy in children.
Otolaryngol Head Neck Surg. 2011;144(1):S1S30.
Schechter MS. Section on Pediatric Pulmonology,
Subcommittee on Obstructive Sleep Apnea
Syndrome. Technical report: diagnosis andmanagement of childhood obstructive sleep
apnea syndrome. Pediatrics, 2002; 109(4): e69.
Patel HH, Straight CE, Lehman EB, Tanner M,
Carr MM. Indications for tonsillectomy: a 10-year

Fiziologia - Physiology • 2020.30.2(100)

14.

15.

retrospective review. Int. J. Pediatr.
Otorhinolaryngol. 2014; 78(12): 2151-2155.
Naja MZ, El-Rajab M, Sidani H, Kabalan W, Ziade
MF, Al-Tannir MA. Modified infiltration techniquein tonsillectomy: expanded case report of 25
children. Int J Pediatr Otorhinolaryngol. 2005; 69:
35-41.
Pierce CA, Voss B. Efficacy and safety of
ibuprofen and acetaminophen in children andadults: a meta-analysis and qualitative review.
Ann. Pharmacother. 2010; 44(3); 489-506.
Star K, Choonara I. How safe is paracetamol?
Arch Dis Child. 2015; 100(1): 73-74.
Michelet D, Andreu-Gallien J, Bensalah T, et al. A
meta-analysis of the use of nonsteroidal
antiinflammatory
drugs
for
pediatric
postoperative pain. Anesth. Analg. 2012; 114(2):393-406.
Jitpakdee T, Mandee S. Strategies for preventing
side effects of systemic opioid in postoperative
pediatric patients. Paediatr. Anaesth. 2014; 24(6):561-568.
Kuehn BM. FDA: No codeine after tonsillectomy
for children. JAMA 2013; 309(11): 1100.
Windfuhr J, Chen Y, Remmert S. Hemorrhage
following tonsillectomy and adenoidectomy in
15,218 patients. Otolaryngol Head Neck Surg.
2005; 132:281-286.
Duval M, Wilkes J, Korgenski K, Srivastava R,
Meier J. Causes, costs, and risk factors forunplanned return visits after adenotonsillectomy
in children. Int J Pediatr Otorhinolaryngol. 2015;
79 (10): 1640-1646.
Uysal HY, Takmaz SA, Yaman F, Baltaci B, Başar
H. The efficacy of intravenous paracetamol
versus tramadol for post-operative analgesia
after adenotonsillectomy in children. J Clin
Anesth. 2011; 23(1):53-7.
Krishna S, Hughes L, Lin S. Postoperative
hemorrhage with nonsteroidal anti-inflammatory
drug use after tonsillectomy: a meta-analysis.
Arch Otolaryngol Head Neck Surg. 2003; 129:1086-1089.
Ozkiris M, Kapusuz Z, Yildirim Y, Saydam L. The
effect of paracetamol, metamizole sodium, andibuprofen on postoperative hemorrhage following
pediatric
tonsillectomy.
Int
J
Ped
Otorhinolaryngol. 2012; 76:1027-1029.
Kelly L, Rieder M, Van den Anker J, et al. Morecodeine fatalities after tonsillectomy in North
American children. Pediatrics. 2012; 129:13431347.

79

DUREREA POST-AMIGDALECTOMIE LAADULŢI – STUDIU COHORTĂREZUMAT
Amigdalectomia este una dintre procedurile comune efectuate cel mai frecvent anual. Durerea este raportată latoți pacienții în perioada post-operatorie și persistă ca simptom pe intervale variabile de timp. Numeroși pacienți
se adresează Serviciului de Urgență după amigdalectomie pentru a primi medicație antialgică. Scopul acestuistudiu a fost de a compara efectul Ibuprofenului vs. Paracetamol vs. Tramadol în perioada post-operatorie derecuperare după amigdalectomie și legătura dintre aceste medicamente și sângerarea post-operatorie
Cuvinte cheie: tonsilectomie, AINS, acetaminophen, tramadol, sângerare

80

Fiziologia - Physiology • 2020.30.2(100)INTERACTIVETEACHINGPEN

DIGITIZERTOFOSTERSAMITA R SORTE1, SACHIN B RATHOD2, KAJAL MOHITE1, PAYALKASAT1, GAYATRI MUTHIYAN1, ASHOK AHIRWAR1, ASHWINI PATIL3
1Department of Physiology, AIIMS Nagpur
2Department of Physiology, IGGMC, Nagpur
3Symbiosis Medical college for Women, Symbiosis International (DU), Pune Maharashtra

ABSTRACT
Background: Blended learning with chalk-board and PowerPoint is the most preferred method of teaching inIndian Medical College. With evolving technology, new digital learning aids are devised to spawn a better teaching
method. Medical teachers keep them updated with newer teaching technologies and tools for better engagementof students in lecture. One such newer teaching aid with advanced technology is “interactive pen digitizer”.
Objectives: To compare the two different teaching tools- “traditional chalkboard” versus “interactive pen digitizer”
from the student’s perspective
Methods: A prospective, cross-sectional, questionnaire-based study was carried out among first-year Indian
MBBS students.
Results: A total of 49 students participated in the study, 33 (67%) participants were males, with a median age of
19.47 years and 16 (33%) participants were females with a median age of 19.39 yrs. 92% of Students preferred“digital pen digitizer as compared to traditional chalk and board.
Conclusions: Students preferred Pen digitizer over a traditional chalkboard for being more fascinating, moreclass-engaging, better summarization, memorization, and focused teaching with fewer distracters.
Keywords: chalkboard, pen digitizer, digital chalkboard, tablet, medical students.

INTRODUCTION
Teaching and learning are active and continuous
processes. Since ancient times, the most common andpreferred large group teaching method is lectures. A
large group method to teach medical physiology ismainly didactics lectures. A good lecture is the mostefficient way to assimilate and impart a large volume
of information from multiple sources in a limited time.
So planning the lecture is a crucial step. It shoulddeliver the content stepwise, logically, utilize the time
wisely, and focus on the important points. A blend ofPowerPoint presentations added with chalkboard
serves this basic motto. Many studies are conductedto compare these two teaching aids and a blend ofboth teaching aids is preferred by medical teachersand students [1-4].

With evolving, technology newer teachingmodalities are introduced. New digital learning aids
are devised for better teaching-learning. To improve
the quality of teaching, medical teachers areprogressively using the latest teaching-learning media
and tools. One such newer teaching aid with advanced
technology is “interactive pen digitizer”. An interactive
pen digitizer consists of a digital pen with a writing padwhich can be connected to the laptop. The size of thewriting pad is 12 inches which are light and easy tohold in one hand while writing. Handwritten notes anddiagrams can be drawn with a digital pen and can bedisplayed on a laptop screen by using software like
“Paint”/ “whiteboard. With pen digitizer, multiple colors
and different tools like pencil, brush or shapes, etc.
can be used. The laptop screen can be projected withthe help of a projector. The matter written on thewriting pad is displayed on the projector in real-time.

Received 14th of October 2020. Accepted 11th of November 2020. Address for correspondence: Dr. Samita R Sorte, AssistantPofessor, Department of Physiology, AIIMS, Nagpur, Plot No. 2, Mihan Khapri, Nagpur Maharashtra, India 441108; phone:+909423409931; e-mail: drsmyta@gmail.com, drsmyta@aiimsnagpur.edu.in

Fiziologia - Physiology • 2020.30.2(100)81Thus pen digitizer is the digital form of Chalk and board
with some added advantage. The impact of Tablet PC[5,6] (similar to pen digitizer) in teaching and learning
has been investigated in many disciplines such asengineering [7-9] mathematics [10] science [11,12]
and education [13,14].

consent was taken from the students who were willingto participate in the study. A total of 49 (n = 49) 1styear medical students participated in the study.
Renal physiology was taught to the students
using the traditional chalkboard method. The
gastrointestinal physiology was taught to the students
using pen digitizer by the same faculty. Aftercompletion of renal and gastrointestinal physiology, apredesigned and pretested 16 item questionnaire
consisting of five sections was given to the students.
The internal consistency of questionnaire was 0.8. The
answers were analyzed and expressed inpercentages.

RESULT
Out of 50 students in the batch, 49 students
participated in the study, 33 (67%) participants weremales, with a median age of 19.47 yrs and 16 (33%)
participants were females with a median age of 19.39
yrs.

Fig. 1. Shows the screenshot of GIT Physiology
taught by using the “digital pen digitizer/Digital Chalkand Board”

Fig. 2. Showing percentages of students preferring
the teaching aids for various questions in section one
and two

There are fewer studies in the medical field [5, 15]
and no studies in Indian Medical Colleges comparing
the traditional “chalk and board” versus “interactive
pen digitizer”. To determine medical student’s
perception comparing the traditional chalkboard withthe pen digitizer, a prospective, cross-sectional,
questionnaire-based study was carried out among
Indian MBBS students.

METHOD AND MATERIAL
The prospective, cross-sectional study was
conducted on 1st-year medical students. Written

82

Fig. 3. Showing percentages of students preferring
Fiziologia - Physiology • 2020.30.2(100)the teaching aids for various question in section threefour and fiveFig. 4. Showing percentages of students preferring
the teaching aids
92% Students preferred digital pen digitizer ascompared to traditional chalk and board.

DISCUSSION
Section one asked the students to assess theirlearning capability with both tools. In this study, weobserve that students were able to perceive the stepby-step sequential development of the concept better
when the topic was taught with pen digitizer. This
allows student’s sufficient time to takes notes. Thesefindings are supported by studies of Frolik et.al (2004)
and Pomales-Garcia. et. al. (2007) and Stickel (2008)[16-19]. In pen digitizer, a good balance between
writing and listening is achieved. Screenshots andscreen recording of the class taught by pen digitizer
can be saved and used to summarize the topic at theend of the class. These notes can be sent or emailed
to the students which can be utilized by students torevise and memorize the topic [16, 20].
Section two addressed the use of multiple colors
for teaching with both teaching tools. Limited color
options are available with chalk and board. Pen
digitizer provided the ability to deliver the traditional
lecture using digital ink [21, 22]. Use of multiple colors
and tools helped to highlights important points andincreased the focus teaching [20, 23-25].
Section three addressed the visibility and clarityof the board. The legibility of handwriting, clarity oftext, and diagram were better with the use of pen
digitizer. Students were able to see the handwritten
notes by pen digitizer on the projector screen from any
corner in the classroom. This has decreased the stresson the eyes. These findings are supported by thestudies done by Derting, et. al. (2008), and Walker et.
al. [26,27]. In open-ended questions students reported
difficulties in seeing the entire blackboard due to glare
Fiziologia - Physiology • 2020.30.2(100)on some corners or reading notes written on theblackboard, distracted them from learning.
Section four asked the question about thestudent’s concentration and distracters. The partial
field of the blackboard is blocked by the teacher
standing in front of the board while writing notes on theblackboard. It blocks the flow of thought anddecreases the speed of notes taking by the students.
It acts as distracters and decreased focus learning.
Teacher-student’s eye contact facilitates thestudents to concentrate on the lecture. When theteacher faces students while teaching, it allows theteacher to track and estimate the student's
comprehension and understanding [28]. But whilewriting on the blackboard the teacher has to face theblackboard. Thus the teacher cannot maintain eye
contact with the students. The students concentrate in
the class slash down and the attention span drives
down in the class. Pen digitizer benefits teachers toface the students while teaching. The potential
technological difficulties which can act as distractersusing pen digitizer were documented in other studies.
These are power cut, computer freezing, slow
computer speed, discharged batteries of the stylus,
etc. [20]. These difficulties were not encountered in ourclasses hence were not reported by our students.
Section five about the student perceptions foroverall satisfaction and effectiveness between two
methods, nearly all students agreed that the pace ofthe lecture was appropriate regardless of the method
used and pen digitizer was the preferred teaching aid.
Students reported greater engagement andgreater satisfaction with Pen digitizer as compared to
traditional chalk and board. Pen digitizer is a promising
tool and does not require a specific teacher training
and preparation. It can switch between otherapplications during lectures and prevents messy chalkand dusty fumes from dry erase markers.
Hong Wang had reports that the use of tablet PCs
with a digital pen or stylus has enhanced students’learning experiences through learner-interface
interaction, learner-content interaction, learner instructor interaction, and learner-learner interaction[29].
Although our study has limitations of small
sample size and was conducted in single-institution, it
provided initial evidence that the Pen digitizer
enhances the effectiveness of didactic lectures. This
teaching aid is preferred by the students. We
recommend the medical teachers should utilize thistechnology for efficient lectures and better student’s
engagement in the class. In the breakneck changing
world, newer educational technology can be an
efficient tool for teaching and learning. It can be acomponent of a comprehensive system for lifelong

83education. Further studies with larger sample size andmulti-centric studies are required to independently
corroborate our data.

12.

REFERENCES
1.

Naqvi SH, Mobasher F, Afzal MAR, Umair M, Kohli AN,
Bukhari MH. Effectiveness of teaching methods in a medical
institute: perceptions of medical students to teaching aids. J
Pak Med Assoc. 2013 Jul;63(7):859-64.
2. Dhaliwal U. A prospective study of medical students’perspective of teaching-learning media: reiterating theimportance of feedback. J Indian Med Assoc. 2007;
105(11):621–3, 636.
3. Novelli ELB, Fernandes AAH. Students’ preferred teaching
techniques for biochemistry in biomedicine and medicine
courses. Biochem Mol Biol Educ. 2007;35(4):263-6.
4. Petimani. Blackboard versus PowerPoint presentation:
Students opinion in medical education, available from:http://www.ijeprjournal.org/article.asp?issn=2395-2296;
year=2015;volume=1;issue=4;spage=289;epage=292;aula
st=Petimani
5. The digitization of medical knowledge: Tablet computerbased medical education and training improves exam
results [Internet]. ScienceDaily. Available from:https://www.sciencedaily.com/releases/2017/05/170504161
520.htm
6. Thomas - A study of effective and best practice usage andi.pdf[Internet].
Availablefrom:https://uhra.herts.ac.uk/bitstream/handle/2299/17553/Thom
as_2016_Effective_and_best_practice_usage_and_implem
entation_of_tablet_PCs.pdf?sequence=2&isAllowed=y
7. John H. Lumkes J. Survey of Three Different Methods ofDelivering Engineering Content in Lectures: Journal of
Educational Technology Systems [Internet]. 2010 May 28;
Availablefrom:https://journals.sagepub.com/doi/10.2190/ET.38.3.e
8. Rogers JW, Cox JR. Integrating a Single Tablet PC in
Chemistry, Engineering, and Physics Courses: Students
Report That an Instructional Model Based on a Single Tablet
PC in Science and Engineering Courses EnhancesClassroom Dynamics, Teaching Effectiveness, and Student
Learning. Journal of College Science Teaching [Internet].
2008Jan
1;37(3):34.
Availablefrom:https://www.questia.com/library/journal/1G1182531026/integrating-a-single-tablet-pc-in-chemistryengineering
9. Hieb JL, Ralston PAS. Tablet PCs in Engineering
Mathematics Courses at the J.B. Speed School ofEngineering. International Journal of Mathematical
Education in Science and Technology. 2010;41(4):487–500.
10. Saber HA. Using maple, maple 3D graphics, and tablet PC
to teach calculus II and III. In: 2016 13th Learning andTechnology Conference (L T). 2016. p. 1–6.
https://www.researchgate.net/publication/314266065_Using_maple_maple_3D_graphics_and_tablet_PC_to_teach_c
alculus_II_and_III
11. Using the Tablet PC for Instruction - Gill - 2007 - Decision
Sciences Journal of Innovative Education - Wiley Online
Library[Internet].
Available
from:

84

13.

14.

15.
16.

17.
18.
19.
20.

21.

22.
23.

24.
25.
26.
27.

https://onlinelibrary.wiley.com/doi/abs/10.1111/j.15404609.2007.00134.x
Pryor G, Bauer V. Building a Better Biology Lab? Testing
Tablet PC Technology in a Core Laboratory Course. Journalof College Science Teaching [Internet]. 2008 Nov 1;38(2):44.
Availablefrom:https://www.questia.com/library/journal/1G1191012914/building-a-better-biology-lab-testing-tablet-pctechnology
Sue Byrd Steinweg, Sarah Carver Williams, Joy Neal
Stapleton. Faculty Use of Tablet PCs in Teacher Education
and K-12 Settings. TECHTRENDS TECH TRENDS 2010;
54(3):54–61.
The Educational Potential of Mobile Computing in the Field[Internet].
Availablefrom:https://er.educause.edu/articles/2011/3/the-educationalpotential-of-mobile-computing-in-the-fieldBaumgart DC, Wende I, Grittner U. Tablet computerenhanced training improves internal medicine exam
performance. PLOS ONE 2017; 12(4):e0172827.
Frolik J, Zurn JB. Evaluation of Tablet PCs for engineeringcontent development and instruction [Internet] 2004.
Availablefrom:https://www.semanticscholar.org/paper/Evaluation-ofTablet-PCs-for-engineering-content-FrolikZurn/322ad1bc55410a7982640e70b7b6a96552a9d072
Pomales‐García C, Liu Y. Excellence in Engineering
Education: Views of Undergraduate Engineering Students.
Journal of Engineering Education, 2007;96(3):253-62.
Stickel M. Effective use of tablet PCs for engineering
mathematics education. In: 2008 38th Annual Frontiers in
Education Conference. 2008. p. S3J-7-S3J-12.
Stickel M, Hum SV. Lessons learned from the first-time useof tablet PCs in the classroom. In: 2008 38th Annual
Frontiers in Education Conference. 2008. p. S1A-7-S1A-12.
Ambikairajah E, Epps J, Ming Sheng, Celler B, Chen P.
Experiences with an electronic whiteboard teachinglaboratory and tablet PC based lecture presentations [DSP
courses]. In: Proceedings (ICASSP ’05) IEEE International
Conference on Acoustics, Speech, and Signal Processing,2005. 2005. p. v/565-v/568 Vol. 5.
Biswas S. Teaching Courses With Tablet Pc: Experience
And Student Feedback. In 2007, p. 12.1351.1-12.1351.9.
Available from: https://peer.asee.org/teaching-courses-withtablet-pc-experience-and-student-feedback
Gill TG. Using the Tablet PC for Instruction. Decision Sci J
Innovative Educ., 2007; 5(1):183-90.
Anderson R, Anderson R, Simon B, Wolfman SA,
VanDeGrift T, Yasuhara K. Experiences with a tablet PC
based lecture presentation system in computer science
courses. SIGCSE Bull. 2004; 36(1):56-60.
Toto R, Wharton M, Cimbala JM, Wise J. One step beyond:Lecturing with a Tablet PC. Computers in Education Journal,
2007; 17(3): 2-11.
Tintarev K, Rydén J. Teaching with digital inkboards.
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.5
57.8589&rep=rep1&type=pdf
Derting TL, Cox JR. Using a Tablet PC To Enhance Student
Engagement and Learning in an Introductory Organic
Chemistry Course. J Chem Educ. 2008; 85(12):1638.
Walker DG, Stremler MA, Johnston J, Bruff D, Brophy SP.
Case study on the Perception of Learning when Tablet PCs
Fiziologia - Physiology • 2020.30.2(100)are used as a Presentation Medium in Engineering
Classrooms.:10.
http://telab.vuse.vanderbilt.edu/docs/walker08IJEE_proof.pdf
28. Whiteboards and Chalkboards | Poorvu Center for Teachingand
Learning[Internet].
Availablefrom:https://poorvucenter.yale.edu/WhiteboardsChalkboards

29. Wang H, Rush BR, Wilkerson M, van der Merwe D.
Exploring the use of tablet PCs in veterinary medical
education: opportunity or obstacle? J Vet Med Educ. 2014;41(2):122-31.

FOLOSIREA STILOULUI DIGITAL INTERACTIV IN
PEDAGOGIEREZUMATÎnvățarea combinată cu tablă și PowerPoint este metoda preferată de predare în Indian Medical College. Cu
tehnologia mereu în evoluție, sunt concepute noi mijloace de învățare digitale pentru a genera o metodă de predaremai bună. Cadrele medicale sunt la curent cu noile tehnologii și instrumente de predare pentru o mai bună implicare
a elevilor în cursuri. Un astfel de ajutor didactic mai nou, cu tehnologie avansată, este stiloul interactiv digital.
Obiective: comparatia intre două instrumente didactice diferite, o tablă tradițională si stiloul interactiv digital, dinperspectiva elevului.
Metode: Un studiu prospectiv, bazat pe chestionar, a fost realizat în rândul studenților din India din anul întâi MBBS.
Rezultate: Un total de 49 de studenți au participat la studiu, 33 (67%) participanți au fost bărbați, cu o vârstă medie
de 19,47 ani și 16 (33%) participanți au fost femei cu o vârstă medie de 19,39 ani. 92% dintre studenți au preferat
stiloul interactiv digital cu stilou în comparație cu creta tradițională și tablă.
Concluzii: Studenții au preferat stiloul interactiv digital unei table tradițională, fiind mai atractiv și permițând o sintetizare
mai bună a cunoștințelor, precum și memorizare mai buna și predare mai concentrată, cu mai puține distracții.
Cuvinte cheie: tablă, stilou digital, tablă digitală, tableta, studenți la medicină.

Fiziologia - Physiology • 2020.30.2(100)85

EVALUATIONOF
THE
POSSIBILITYOF
POSTPARTUM HEMORRHAGE IN PATIENTS WITH
PLACENTAL STRUCTURE ABNORMALITIESDRAGOȘ CRISTIAN CARAVEȚEANU1, DAN-VASILE MOROȘANU1, ANCADANIELA BRĂILA1, MIHAI BRĂILA1
1University of Medicine and Pharmacy of Craiova, Romania

Abstract
Obstetric hemorrhage is associated with an increased risk of severe maternal morbidity and mortality. Postpartum
hemorrhage is the most common form of obstetric hemorrhage, and worldwide, a woman dies from massive
postpartum hemorrhage about every 4 minutes.
In addition, after hemorrhage, many women experience severe morbidity, such as multiple organ failure,complications of multiple blood transfusions, peripartum hysterectomy, and unintentional pelvic organ damage,loss of fertility, and psychological sequelae, including posttraumatic stress disorder.
In this context, I consider useful a study to analyze the risk factors that were correlated with severe postpartum
hemorrhage in probably the most common placental pathology associated with obstetric hemorrhagic syndromes,
namely, placenta praevia, trying to establish a model for predicting postpartum severe hemorrhage., derived fromclinical and imaging results, to guide obstetricians to accurately assess severe bleeding and to prepare treatment
strategies for patients with severe postpartum hemorrhage.
Keywords: obstetric hemorrhage, postpartum hemorrhage, placenta praevia

INTRODUCTION
Anticipating massive postpartum hemorrhage, prompt
recognition of the cause and the establishment ofcorrect and appropriate measures to control bleeding[1,2] and replace lost blood volume and restore
oxygen transport capacity (normal hemoglobin values)and correct the "washing phenomenon" / " washout
phenomenon” that leads to coagulopathies will help
save lives [3,4].
The obstetric shock index can help avoid
underestimating blood loss and the use of tranexamic
acid, oxytocics but also peripartum hysterectomy,
performed in a timely manner, if and whereappropriate, will help save lives [5,6,7].
Obstetric hemorrhage may occur before or after birth,but over 80% of cases occur postpartum [8,9,10].
Globally, massive obstetric hemorrhages, resulting
from the failure of normal obstetric, surgical and / orsystemic hemostasis, are responsible for at least 25%of the several hundred thousand maternal deaths eachyear [11,12].
Most women will not have identifiable risk factors[13,14,15]. However, the primary prevention of
postpartum hemorrhage begins with an assessment ofidentifiable risk factors [16,17,18].
In this context, I consider useful a study to analyze therisk factors that were correlated with severe
postpartum hemorrhage in probably the most common
placental pathology associated with obstetrichemorrhagic syndromes, namely placenta praevia,
trying to establish a model for predicting severe
hemorrhage. postpartum, derived from clinical andimaging results, to guide obstetricians to accurately
assess severe bleeding and to prepare treatment
strategies for patients with severe postpartum
hemorrhage [19,20].

Received 15th of October 2020. Accepted 13th of November 2020. Address for correspondence: Dragos Cristian Caraveteanu,
University of Medicine and Pharmacy of Craiova, Romania, Petru Rares Street no.2, Craiova; phone: 0722160462; email:cristicaraveteanu@yahoo.com

86

Fiziologia - Physiology • 2020.30.2(100)AIM AND OBJECTIVES
The impact on pregnancy in cases of placenta praevia
is most likely reflected in both mother and fetus, eachof which can frequently suffer severe complications
due to it, especially increasing the risk of obstetrichemorrhage[5]. In addition, after hemorrhage, many
women experience severe morbidity, such as multiple
organ failure, complications of multiple bloodtransfusions, peripartum hysterectomy, andunintentional pelvic organ damage, loss of fertility, andpsychological sequelae, including posttraumaticstress disorder [9].
In this research we set several specific objectives,
which would allow the achievement of the initial
proposed goal and the subsequent elaboration of
conclusions derived from the results obtained from thestudy, with a high degree of objectivity andrepresentativeness. Having as origin the desideratum
evoked previously, we proposed that in this study tocarry out in a first stage a clinical study, in order toinvestigate the existence of correlations regarding theinfluence of insertion and placental structure on
pregnancy in general and on obstetrical hemorrhagic
syndrome.
At a later stage of the research we considered it an
objective to study the impact of clinical situations
associated with obstetric hemorrhage, by carefulanalysis and correlation of these data withultrasonography
study
and
macroscopic
morphological study of placenta. Finally, I proposed acomprehensive and integrated approach to theseobjectives, in order to generate a broad perspective onmaternal-fetal management in the context of obstetrichemorrhagic syndrome associated with placenta
praevia, in order to generate the main objective of thisstudy, namely the generation of a model to be able topredict severe postpartum hemorrhage, a model
derived from clinical and imaging information.

MATERIAL AND METHODS
We conducted our research study, performed both
retrospectively and prospectively, between 2014 and2019, on a study group consisting of 82 selected
pregnant patients diagnosed with placenta praevia.
The cases included in the study groups were selectedfrom the case studies of the Obstetrics-Gynecology
Department of the "Caritas" Hospital Roșiori de Vede,the private practice of the Obstetrics-Gynecology
office "Dr. Caravețeanu Dragoș Cristian" and private
practice of 2 other Obstetrics-Gynecology offices in
Teleorman County. This study was conducted entirelyin accordance with existing regulations in the field of
scientific research, provided in both European and
Fiziologia - Physiology • 2020.30.2(100)Romanian legislation but also according to the rules of
the University of Medicine and Pharmacy of Craiova
and by approving the Ethics Commission of the
Senate of the University of Pharmacy from Craiova.
Blood loss was assessed using a combination ofgauze weighing methods and blood clot collection,
based on the postpartum hemorrhage management
and prevention protocol issued by the Obstetrics andGynecology Associations of Europe and Romania,
was measured at the beginning of the skin incision for
cesarean section or at the beginning of perioperative
vaginal bleeding for cesarean section or at the
beginning of parturition for vaginal birth and blood loss
over 1,000 ml was defined as severe postpartum
hemorrhage and was the only clinical observation inthis study. The diagnosis for placenta praevia wasmade after 28 weeks of pregnancy using
ultrasonography. Clinical suspicion of placenta
praevia was maintained in the following cases:
pregnant patient with vaginal bleeding at a minimum
time after 24 weeks of amenorrhea, unprovokedassociated with the absence of painful uterine
contractions, with a normal uterine tone present, withoo dystocic presentation, with fetal heartbeats present
and normal. In order to better outline the diagnosis, weassociated the following associated conditions:intrauterine growth retardation; the presence ofmaternal risk factors, including a history of placenta
praevia; premature rupture of membranes.
Based on the guidelines and recommendations
published by the profile associations, we collectedclinical information regarding postpartum hemorrhage,noting the following aspects: age; residence; level of
education; assisted reproduction; pregnancy history;
number of tasks; hours of birth; periods of miscarriage;
history of vaginal birth; history of cesarean section;
history of uterine surgery; history of surgicalcomplications; history of premature births; history ofplacenta praevia; current information about the task;
body mass index before pregnancy; number ofpregnancy tests; the presence of uterine fibroids; thepresence of anemia; the presence ofthrombocytopenia; diagnosis of hypertension;
amniotic fluid index at the last ultrasound; history ofvaginal bleeding during pregnancy; bleeding volume;
possible treatment with magnesium sulfate; fetal
cephalic presentation; gestational weeks attermination of pregnancy; surgical anesthesia; vaginal
blood loss; blood transfusion.
In developing the model for predicting the possibility ofobstetric hemorrhage and given the case of theexistence of medical units in disadvantaged areas,
which do not have adequate ultrasonographyequipment or in the case of massive hemorrhage,
which do not allow the patient to move immediately to87

a clinic benefit from such a facility, the formulation of
the diagnosis of placenta praevia was also used basedon the specialized clinical examination, obstetrical andespecially the vaginal one.
Based on the guidelines and recommendations
published by the profile associations, we collected
information about the placenta related to postpartum
hemorrhage, including the type of placenta: lower
inserted placenta, the lowest location of the placenta
at 2 cm from the internal cervical orifice; marginalplacenta: the placenta that reaches the internalcervical orifice; partial placenta: part of the placentathat covers the internal cervical orifice; complete
placenta: placenta that completely covers the internalcervical orifice); place of the placenta (the main bodyof the placenta that covers the anterior wall of theuterus, defined as the anterior wall of the placenta andthe one that covers the posterior or lateral wall of theuterus defined as the posterior wall of the placenta);echo of the uterine placenta junction (spongy placenta;
appearance of placental fissure: 4 or more regionswith reduced echo, with a diameter greater than 5 mmin the placenta); the placenta covering the uterinebladder junction (the uterine serous line is clear andcontinuous, the uterine serous line is discontinuous or
not completely visible); abundance of blood vessels at
the junction of the uterine placenta assessed under
Color Doppler control.
Depth of the placenta invading the wall of the uterinemuscle: based on the richness of vascularity at theecho of the junction of the uterine placenta, thejunction of the uterine bladder covered by the placenta
and the junction of the uterine placenta. The
correlation was preliminarily assessed based onabnormal implantation, placental adhesion, placental
implantation and placental penetration.
Statistical processing of results and comparative data
analysis was performed using the following softwares:
Microsoft Excel version 2007, Anova One Way,
respectively SPSS version 22.0. Sublots analyze was
performed by statistical indicators: weight, median,standard deviation. Testing the consistency of thesamples was performed using the independent
Student t-test, bilateral, and testing the combination ofparameters by using the Chi square test of
association. Statistical differences of the test wererepresentative when the p value was <0.05 at aconfidence interval of 95%.

88

RESULTS
Our results shows that from the total of 82 patientsenrolled in the study, a number of 6 had a vaginal birth,while a number of 76 pregnant women included in the
study group, gave birth by cesarean section. Analyzing
the data that characterize the main characteristics ofthe study group, it is observed a predominance of
patients from urban areas, namely 61 from the study
group, the mean age was 31 years, the duration ofgestation was in most patients included in the study
under 39 weeks (over 90%) and only a number of 8
patients had over 39 weeks of pregnancy.
We assessed collateral pathology, identifying 6
patients with high blood pressure during pregnancy,
14 patients with preeclampsia, 1 patient presented
high values of fasting blood glucose and subsequently,
by consultation in the outpatient clinic, was diagnosed
with gestational diabetes. Nevertheless we identified
obesity in a number of 13 patients, the presence ofabortion in the antecedents (13 patients), 4 patientsfrom the studied group (representing 4.88% of thetotal) reported the loss of a pregnancy, while 1 patientfrom the studied group (representing 1.22% of thetotal) evoked the loss of two previous pregnancies.
From the personal pathological antecedents, it wasalso highlighted that 2 patients from the studied group(representing 2.44% of the total) had venous
thrombosis.
On the other hand, out of a total of 82 patients enrolled
in the study, a percentage of 7.31% from the study
group had a vaginal birth, while the rest gave birth bycesarean section (all of these patients were initiallydiagnosed with the lower placenta inserted).
The most important findings was reporting postpartum
hemorrhages All the 82 patients included in our grouppresented postpartum hemorrhages, including a
number of 21 patients, representing a percentage of
26% having severe hemorrhages, over 1000 ml, while
a number of 61 patients, representing a percentage ofthe studied group of 74% had minor bleeding.
Regarding the age of pregnant patients included in the
study group, in the case of patients with severe
postpartum hemorrhage, the mean age was,according to statistics, about 32 years, with extremesof 23 years and 41 years, respectively, in while the ageof pregnant patients included in the study group who
had minor postpartum hemorrhage, the mean age wasapproximately 29 years, with extremes of 19 years and38 years, respectively. There is a difference of about
3 years in favor of patients who had severe bleeding,the difference being maintained even in patients who
had the extreme ages of the group.
Also, a number of 59 patients, of all those who had
minor postpartum hemorrhage (61 patients) had agestation period of less than 38 weeks of pregnancy,
Fiziologia - Physiology • 2020.30.2(100)while in the case of pregnant patients included in the
study group who had severe postpartum hemorrhage,
90.48% of the total had a gestation period of lessthan 38 weeks of pregnancy.
We report a slight difference of about 6 percent in favorof patients with minor bleeding, but without a strong
statistical significance and a higher percentage infavor of patients born by caesarean section and had
major postpartum hemorrhage.
In the statistical calculations we also took into accounta whole series of other clinical indices in the prediction
model of postpartum hemorrhages, among which
history of premature birth 2 patients with minor
hemorrhage, and a patient with severe hemorrhage,history of placenta praevia, one patient with severe
hemorrhage, the presence of uterine fibroids one
patient with severe hemorrhage.
We also observed a higher percentage in favor ofpatients born by caesarean section and had major
postpartum hemorrhage, both to patients with naturalbirth and major postpartum hemorrhage, and to bothsubcategories (natural birth and cesarean section)
within patients with minor bleeding studied. According
to clinical evaluations, a number of 32 patients withminor hemorrhage, representing a percentage of
52.46%, had anemia in pregnancy. Regarding thepatients with severe hemorrhage, included in thestudied group, anemia during pregnancy wasdiagnosed in a number of 11 patients, but representing
a percentage of 52.38%. Of all the patients included in
the study group, a number of 9 patients had a historyof bleeding in the last trimester of pregnancy, of which
6 patients, representing a percentage of 9.84%
included in the subgroup with minor hemorrhage, while
3 patients, representing a percentage of 14.29%
included in the subgroup with severe hemorrhage,
having a statistically significant higher percentage.
The primary factor we reported in this study was, as it
results from the formulation of the working hypothesis
and the establishment of batches, the volume of
postpartum hemorrhage, which was calculated
according to the algorithm presented in the chapter
Material and methods. From the evaluations ofobstetricians and doctors of anesthesia and intensive
care, the following were observed: the patients withminor hemorrhage included in the studied grouppresented an average value of blood loss of 647 mlwith extremes between 367 ml and 927 ml
respectively; the patients with severe hemorrhage
included in the studied group presented an averagevalue of blood loss of 2145 ml with extremes between
1346 ml and 3332 ml.
From the point of view of the type of placenta,
evaluated by ultrasonography and according to theclassifications taken in the clack, the patients
Fiziologia - Physiology • 2020.30.2(100)considered in the study group presented: lower
inserted placenta - 17 patients with minor hemorrhage,
one patient with severe hemorrhage; marginal praevia
placenta: 4 patients with minor hemorrhage, onepatient with severe hemorrhage; partial praevia
placenta - one patient for each, minor and severe
hemorrhage; central placenta praevia - 39 patientswith minor hemorrhage, respectively 18 patients withsevere hemorrhage.
Thus, patients with minor hemorrhages presented a
distribution of the placenta type as follows: 27.87% lower inserted placenta, 6.56% - marginal praevia
placenta, 1.64% - partial praevia placenta, 63.93% praevia placenta central. Also, the patients with severe
hemorrhages presented a slightly different distribution,
but keeping the hierarchy of the placenta type asfollows: 4.76% - lower inserted placenta, 4.76% marginal praevia placenta, 4.76% - partial praevia
placenta and the vast majority 85.72% - central
placenta praevia.

DISCUSSION
Logical stepwise regression analysis of risk factors
indicated that age, duration of birth, history of vaginal
birth or cesarean section, weeks of pregnancy, depthof implantation of the placenta invading the uterinemuscle wall, and blood transfusion during birth wererisk factors, independent for severe postpartum
hemorrhage.
We found that blood transfusions during birth were an
intraoperative variable and should be predetermined
before birth when variables are included to determine
the pattern of bleeding. Therefore, it should be omitted
when establishing the model. The logical regression
prediction model was used to predict severe
postpartum hemorrhage after random sampling using
the R language, setting the discriminant limit value ofthe model, with a probability between 0 and 1 (0.3). In
other words, after entering each variable in the model
equation, if the calculated probability is greater than0.3, it denotes a higher occurrence of severe
postpartum hemorrhage, while a value lower than 0.3
denotes a lower probability.
The equation of the P prediction model of the logistic
risk was as follows:
P = Log (F / 1 - F), where F (hemorrhage factor) = −6.9
+ 0.075 x V (age) + 1.5 x P (first birth = 1, multiplied by
the number of pregnancies) - 3 .5 x N (vaginal birth: 1for yes and 0 for no) + 1.7 x N (duration of pregnancy- 0 for <38 weeks, 1 for ≥38 weeks) + 1.5 x D(relationship between placenta and wall uterinemuscle: 0 for normal, 1 for placental adhesion, 2 forplacental implantation, 3 for placental penetration) +0.2 x A (miscarriage = 1, no = 0).
89

For example, a 30-year-old pregnant woman (V = 30),
who gave birth once (P = 1), vaginal birth 0 (V = 0),
gestational weeks at termination of pregnancy 37 (N =0), normal in the placenta (P = 0), miscarriage once (A= 1) an F value of: F = −6.9 + 0.075 × 30 + 1.5 × 1 3.5 × 0 + 1.7 × 0 + 1.5 × 0 + 0.2 x 1 = −2.95, F wasintroduced into the equation logic to obtain P = 0.0434<0.3, indicating a lower chance of severe postpartum
hemorrhage.
To verify the effectiveness of the logistic risk prediction
model equation, 15 patients were randomly selectedfrom the total (n = 82) to determine the verification rate.
The results showed that the sensitivity was = 0.73
(negative prediction accuracy rate), the specificity was= 0.9 (positive prediction accuracy rate), and the total
accuracy rate was 0.8.

CONCLUSION
The established model we generated predicted severe
postpartum hemorrhage in patients with placenta
praevia based on independent risk factors for severe
postpartum hemorrhage and included clinical andultrasonography data. The sensitivity and specificity ofthe model was 73% and 90%, respectively.
Obstetricians could effectively use this model todetermine the risk of severe postpartum hemorrhage
in patients with placenta praevia.

REFERENCES
1.
World Health Organization (WHO). Maternalmortality,
15
March
2016,
http://www.who.int/mediacentre/factsheets/
fs348/en/(accessed 24 October 2016).
2.
Say L, Chou D, Gemmill A, et al. Global causes ofmaternal death: a WHO systematic analysis. Lancet Glob
Health2014;
2(6):e323–e333,
http://www.thelancet.com/journals/langlo/article/PIIS2214109X(14)70227-X/fulltext.
3.
Saving Lives Improving Mothers’ Care: Surveillanceof maternal deaths in the UK 2011-13 and lessons learned to
inform maternity care from the UK and Ireland Confidential
Enquiries into Maternal Deaths and Morbidity 2009-13.
MBRRACE-UK, December 2015, https://www.npeu.ox.ac.
uk/downloads/files/mbrrace-uk/reports/MBRRACE-UK%20
Maternal%20Report%202015.pdf
4.
Mavrides E, Allard S, Chandraharan E, et al.
Prevention and management of postpartum haemorrhage.
BJOG 2016; 124: e106–e149.
5. NCCEMD, Saving Mothers 2005–2007: Fourth Report on
Confidential Enquiries into Maternal Deaths in South Africa.

90

Pretoria, SA: South African Department of Health, 2009: 11–47.
6. ACOG Practice Bulletin: Clinical Management Guidelines for
Obstetrician-Gynecologists Number 76, October 2006:
postpartum hemorrhage. Obstet Gynecol 2006; 108: 1039–
47.
7. Khan KS, Wojdyla D, Say L, Gulmezoglu AM, van Look PF.
WHO analysis of causes of maternal death: a systematic
review. Lancet 2006; 367: 1066–74.
8. Hogan MC, Foreman KJ, Naghavi M, et al. Maternal mortality
for 181 countries, 1980–2008: a systematic analysis ofprogress towards Millennium Development Goal 5. Lancet
2010; 375: 1609–23.
9. Knight M, Callaghan WM, Berg C, et al. Trends in postpartum
hemorrhage in high resource countries: a review andrecommendations from the International Postpartum
Hemorrhage Collaborative Group. BMC Pregnancy Childbirth2009; 9: 55.
10. McCormick ML, Sanghvi HC, Kinzie B, et al. Preventing
postpartum hemorrhage in low-resource settings. Int J
Gynaecol Obstet 2002; 77: 267–75.
11. Geller SE, Adams MG, Kelly PJ, et al. Postpartum
hemorrhage in resource-poor settings. Int J Gynaecol Obstet
2006; 92: 202–11.
12. Miller S, Lester F, Hensleigh P. Prevention and treatment of
postpartum hemorrhage: new advances for low-resource
settings. J Midwifery Womens Health 2004; 49: 283–92.
13. Bose P, Regan F, Paterson-Brown S. Improving theaccuracy of estimated blood loss at obstetric haemorrhage
using clinical reconstructions. BJOG 2006; 113: 919–24.
14. Francois KE, Foley MR. Antepartum and postpartum
hemorrhage. In:Gabbe S, Niebyl J, Simpson J, eds.
Obstetrics: Normal and Problem Pregnancies, 5th edn.
Philadelphia, PA: Churchill Livingstone Elsevier, 2007: 456–
85.
15. Macphail S, Fitzgerald J. Massive post-partum
haemorrhage. Curr Obstet Gynaecol 2001; 11: 108–14.
16. Konje JC, Murphy P, de Chazal R, et al. Severe factor X
deficiency and successful pregnancy. Br J Obstet Gynaecol
1994; 101: 910–1197. Jansen C, de Mooij YM, Blomaard CM,
Derks JB, van Leeuwen E, Limpens J, Schuit E, Mol BW,
Pajkrt E. Vaginal delivery in women with a low-lying placenta:a systematic review and meta-analysis. BJOG. 2019
Aug;126(9):1118-1126.
17. Matsuzaki S, Kimura T. Vasa Praevia. N. Engl. J. Med. 2019
Jan 17;380(3):274.
18. Sanad AS, Mahran AE, Aboulfotouh ME, et al. The effect ofuterine artery ligation in patients with central placenta pevia:
a randomized controlled trial. BMC Pregnancy Childbirth.
2018 Aug 29;18(1):351.
19. Committee on Practice Bulletins-Obstetrics. Practice
Bulletin No. 183: Postpartum Hemorrhage. Obstet Gynecol.
2017 Oct;130(4):e168-e186.
20. Pedigo R. First trimester pregnancy emergencies:
recognition and management. Emerg Med Pract. 2019
Jan;21(1):1-20

Fiziologia - Physiology • 2020.30.2(100)EVALUAREA POSIBILITĂȚII DE HEMORAGIE POSTPARTUM LA PACIENȚII CU ANOMALII DE
STRUCTURĂ PLACENTARĂ
RezumatHemoragia obstetricală este asociată cu un risc crescut de morbiditate și mortalitate maternă severă. Hemoragia
postpartum este cea mai frecventă formă de hemoragie obstetrică și, la nivel mondial, o femeie moare din cauza unei
hemoragii masive postpartum aproximativ la fiecare 4 minute.
În plus, după hemoragii, multe femei suferă de morbiditate severă, cum ar fi insuficiența multiplă a organelor, complicațiile
transfuzionale multiple, histerectomie totală și afectarea involuntară a organelor pelvine, pierderea fertilității și sechelele
psihologice, inclusiv tulburarea de stres posttraumatic.
În acest context, considerăm util un studiu pentru a analiza factorii de risc care au fost corelați cu hemoragia severă
postpartum în probabil cea mai frecventă patologie placentară asociată cu sindroamele hemoragice obstetricale și anume
placenta praevia, încercând să stabilim un model de previziune a hemoragiei severe postpartum, derivat din rezultatele
clinice și imagistice, pentru a ghida obstetricienii să evalueze cu precizie sângerările severe și să pregătească strategii detratament pentru pacientele cu hemoragie severă postpartum.
Cuvinte cheie: hemoragie obstetricală, hemoragie postpartum, placenta praevia

Fiziologia - Physiology • 2020.30.2(100)91

DEVELOPMENT OF A MINIATURIZED METHOD FOR
ESTIMATING
ARISTOLOHIC
ACID
I
(AAI)
IN
ENVIRONMENTAL SAMPLESLAVINIA PAULA DRĂGHIA1,2, ALEXANDRA TEODORA LUKINICH-GRUIA2,
CSILLA ZAMBORI1,2, CARMEN TATU1,2, GABRIELA TĂNASIE1,2, CĂLINADRIAN TATU1,2, VIRGIL PĂUNESCU1,2, PILAR CAMPÍNS-FALCÓ3, YOLANDA
MOLINER-MARTÍNEZ3
1Immuno-Physiology and Biotechnology Center, Department III of Functional Sciences, University of Medicine and Pharmacy 'Victor Babeș'Timișoara, Eftimie Murgu Sq. 2, Timisoara, RO-300041, Romania
2OncoGen Centre, County Hospital 'Pius Branzeu', Blvd. Liviu Rebreanu 156, RO-300736, Timisoara, Romania
3Departamento Química Analítica, Facultad de Química, Universidad de Valencia, Dr. Moliner 50, Burjassot, 46100 Valencia, Spain

ABSTRACT
Aristolochic acid I (AAI) is known as the principal poisonous nitrophenanthrenic compound belonging to the big groupof aristolochic acids (AAs), naturally produced by the Aristolochia and Asarum genus.This phytotoxin is considered themain causative factor involved in Balkan endemic nephropathy (BEN), a deadly environmentally-induced chronic
kidney disease present in particular Balkan locations, including few villages from Romania. In this article, we highlighted
various types of extraction techniques used for AAI detection and quantification in different environmental samples,developed for specific chromatography methods. In order to extend the scope of our previous studies regarding theoccurrence and distribution of AAI contamination in "soil-plant-environment" systems, we proposed a novel sensitive
method of miniaturized extraction techniques: in-tube solid-phase microextraction (IT-SPME) coupled with capillaryliquid cromatography (Cap-LC). Chromatographic separation and quantification of AAI were carried out under isocratic
conditions, using a particulate LC column (Zorbax SB-C18) and DAD connected to the capillary column (TRB-35) bymeans of a switching valve. Linearity ranged between 10–100 ng/ml, achieving a limit of detection (LOD) of 8.12 ng/mL.
The proposed method is cost effective to determine AAI in environmental samples, has good analytical characteristics
and allows the identification and quantification of AAI in appropriate concentrations to control environmental quality.
The characterization of samples can be achieved in less than 10 minutes.
Keywords: Aristolochic acid I; Balkan endemic nephropathy; Environmental samples; Extraction; Liquid
chromatography

1. INTRODUCTION
Data collected over the past sixty years indicate thatcertain rural villagers from the Balkan countries along the
Danube River (Romania, Serbia, Bulgaria, Bosnia andCroatia) are affected by a particular renal disease known
as Balkan endemic nephropathy (BEN). The mostdistinguishing features of BEN are: its endemic nature,affecting residents from certain rural farming villages in the
Balkan area, long-incubation period and a familial, but notinherited, pattern of disease; occurrence in adults but not

children; and a close association with carcinomas of theupper urinary tract (UTUC) [1-7].
BEN is a unique type of gradual chronic renal
disease, with arguable etiology, but throughout the timeseveral factors have been speculated: Pliocene lignite,heavy metals, fungal toxins and polycyclic aromatichydrocarbons (PAHs) without any concluding results [1-5].
In 1969 Ivic was the first to propose another etiologic
factor, aristolochic acids (AAs), coming out from thewidespread toxic Aristolochia plants and therefore
entering in contact with the food around and contaminating

Received 14th of September 2020. Accepted 13th of October 2020. Address for correspondence: Draghia Lavinia, PhD student,
Immuno-Physiology and Biotechnology Center, Department III of Functional Sciences, University of Medicine and Pharmacy'Victor Babeș' Timișoara, Eftimie Murgu Sq. 2, Timisoara, RO-300041, Romania; OncoGen-Centre for Gene and Cellular Therapies
in the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency County Hospital Timisoara, Romania, Liviu Rebreanu No.156 Blv.,
Timisoara, RO-300723, Romania; phone/fax: +40726307304; e-mail: draghia_lavinia@yahoo.com

92

Fiziologia - Physiology • 2020.30.1(99)it [8]. The nephrotoxicity of the AAs became clear in the
1990s, in Belgium, when over 100 young consumed
slimming herbal preparations made from Aristolochia
species and accused symptoms of renal disease [9,10].
This rapidly progressive kidney disease was originallynamed Chinese herb nephropathy (CHN) and later
renamed aristolochic acid nephropathy (AAN), having
similar clinical and morphological parameters. [11].
Aristolochic acids (AAs) are a group of
nitrophenanthrene carboxylic acids, naturally found in the
Aristolochia species, classified as Group 1 carcinogens by
the International Agency for Research on Cancer (IARC),
therefore some countries banned the commercialization of
AA-containing herbal medicines [12].
In the past ten years, different groups of researchers
further proposed and consolidated the hypothesis that
BEN is an environmentally induced disease bydemonstrating the toxicity of AAs and their occurrence infood and the environment [13-15]. Environmental samples
such as: soils, common vegetables and drinking water
were analyzed and confirmed the existence of AAs in rural
food crops around the endemic BEN areas or where
Aristolochia clematitis grows [16-23]. As a result AAscontaining weed comingling with the food crops around(cucumber, maize, grain, wheat) [16,17] was found to be
responsible of environmental exposure AAs and chronic
dietary poisoning [14-23].
Due to the high toxicity, the quantification of AAs has
an obvious significance for the prevention of adverse
events through early detection. In recent years, a numberof methodologies have been developed for the detection
and quantification of AAs extracts [24].
Although many articles have been published on this
topic, the separation efficacy needs to be further improved,
especially for the analysis of real samples. Most of the
proposed methods are used to quantify AAs in herbal
remedies, which contain relatively high concentrations ofAAs, but they may not be sufficiently sensitive to detect
low levels of AAs in the complex matrices of food andenvironmental samples [25].
BEN represents a public health problem, so there isan increasing interest to develop a simple and sensitive
detection method for emerging pollutants like aristolochic
acids (AAs) that are affecting sporadic populations in the
Balkan area. Aristolochia clematitis plants are considered
the main cause of BEN, so controlling the growth of thisweed, together with prevention and remediation methods
would necessary to fight this deadly disease [26].
Nowadays there is a general trend in analyticalmethods towards simplification and miniaturization of thesample preparation process, which reduce the sample
handling and analysis time, as well as the consumption oflarge volumes of solvents and the generation of wastes. In
this sense, in-valve in-tube solid phase microextraction(IT-SPME) has emerged as a reliable technique for the
online enrichment of analytes from environmental samples
Fiziologia - Physiology • 2020.30.2(100)[27-29]. Therefore, in this study we are proposing a new
minuaturized extraction technique in order to achieve good
separation and sensitivity for the determination of AAI inenvironmental samples.

2. MATERIALS AND METHODS
2.1 Reagents and materials
All chemicals and reagents were of analytical quality
and were used without further purification.The standard
AAI was purchaised from Sigma-Aldrich (St. Louis,
Missouri,USA). The standard solution of AAI was preparedin methanol at the concentration of 70 mg/mL. Working
solutions of AAI were obtained by dilution of the stock
solutions with water. All solutions were stored in amberglass bottles at 4◦C. Methanol, acetonitrile and formic acid
were purchased from Merck (Darmstadt, Germany). Highpurity water was prepared using a Milli-Q water purification
system (Millipore, Milford, MA, USA).
2.2 Instrumental analysis & Chromatographic conditions
The capillary chromatographic system consisted of a
LC isocratic capillary pump (Agilent) and a UV diode array
detector (Agilent 1260, Palo Alto, CA, USA) with six-port
injection valveequipped with a GC capillary column(TeknokromaTRB-35) which acted as the injection loop.
The detector was linked to a data system (Agilent, HPLC
ChemStation) for data acquisition and calculation. The
Zorbax SB-C18 analytical column (150 mm x 0.5 mm i. d.,5 um) purchased from Agilent was used for the separationof the analyte (AAI). The column was eluted with aisocratic binary solvent system consisting of
acetonitrile:water (50:50) plus 0.1% formic acid, at a flowrate of 20 uL/min. The excitation and emission
wavelengths of the UV detector were set at 315 nm.
2.3 Extraction techniques used for aristolochic acid I(AAI)
2.3.1 Solvent extraction
In order to efficiently extract AAI, several factors haveto be considered in this process, starting with the sample
preparation. A pre-cleaning step of the samples before theextraction process would be essential to reduce
interactions with other compounds [30-32]. The extraction
method should be non-selective in order to avoid
destroying the structure of the chemical compounds. Fromall extraction methods, solvent extraction is the mostwidely used and methanol is most commonly reported to
be used in AAs extractions [33-35]. The yield of AA
extraction depends on the type of solvents, the volume ofthe solvent, the extraction time and the temperature.
Among the solvents with variable polarities, the highest
yields of AAs were obtained with 70% methanol in93aqueous medium. Extraction conditions were optimized byinvestigating the efficiency of different extraction methods(ultrasound versus reflux). Sonication with methanol, amethod often reported in the literature, has proven to be
simple and effective [31-36].
The analysis and quantification of AAs can be doneby developing a method that takes into account theproperties of AAs but also the matrix, which can be a raw
extract, medicinal preparation, herbal supplement or otherenvironmental sample. Therefore, over the past years,
several extraction methods have been proposed [37]. In
our previous studies, we employed different
chromatography methods (HPLC, LC-MS and GC-MS) in
order to analyze environmental samples (soils andvegetables)
For the case of HPLC and UHPLC-MS analysis, weemployed a simple extraction method adapted after
Trujillo et al. [36] where we mixed 5 grams of sample with
3 mL extraction solvent, which consists of: 80% methanoland 20% aqueous solution with 10% formic acid. The
solid-liquid mixture was centrifuged at 4000 rpm for 10
minutes and then the supernatant was collected andfilteredthrougha0.45 μm
polyethersulfone(PES) filter before injection into HPLC and UHPLC-MS[19].
Another extraction technique used for GC-MS
analysis involves 1:1 ratio of weight of sample:volume of
methanol. After combining the two components, we
obtained a mixture that was heated to 350 Watts in a
microwave oven in two rounds of 30 seconds. In order toseparate the extract, the samples were centrifuged at2000 rpm for 10 min, then the supernatant was collected
and evaporated to a continuous stream of nitrogen. The

3. RESULTS AND DISCUSSION
3.1 Method calibration and validation
The standard AAI solution was prepared inmethanol at a concentration of 70 μg/mL. AAI working
solutions were obtained by diluting the stock solutions with
distilled water. All solutions were stored in amber glass
vials at 4°C.
Detection and quantification of AAI was possible forall extracted samples, which were analyzed by Cap-LC
method with UV detection, a calibration curve with serial
dilutions was performed daily, in the range of94

samples were resuspended in 20 µL methanol andvortexed to obtain a homogeneous content and theninjected into the GC-MS [38].
2.3.2 IT-SPME extraction
IT-SPME(In-Tube
Solid
Phase
Microextraction) extraction technique was carried out
using a capillary column of the type TRB- GC 35(Teknokroma, Barcelona, Spain) with the length ranging
from 40 to 70 cm, covered with 35% diphenyl and 65%
polydimethylsiloxane (3 µm coating and 97 µm thickness)connected in a six-port injection valve, replacing theinjection loop. Capillary connections to the valve were
facilitated by the use of 2.5 cm sleeve of 1/16 in polyether
ether ketone (PEEK) tubing and ferrules (Teknokroma,Barcelona, Spain).
Following preliminary studies, the volume ofprocessed samples was optimized. For this aim, we tested
volumes between 25-1500 µL. In the optimized
procedure, aliquots of 500 µL standards and samples
were injected manually using a high-precision syringe(Hamilton, Bonaduz, Switzerland) a volume of 500 µL into
the extraction column of the IT-SPME device. Finally, theinjection valve was rotated manually, so the analyzes
were transferred directly to the LC system through thecapillary analytical column, so that extraction,
preconcentration, separation and detection are performed
in a single step, using dynamic desorption. The mobile
phase is passed through the capillary column and thedesorbed analytes are sent to the injection valves of thechromatographic system, following the normal circuit of
the Cap-LC system [27].

concentrations 10 - 100 ng/mL. The AAI quantification was
based on several key parameters obtained from thecalibration curve. Calibration curves for AAI were linear in
the concentration test range and were generated bygraphically representing the area of AAI peaks relative to
AAI concentrations of working standards (Figure 1). The
detection limit (LOD) was calculated at 8.12 ng/mL,
obtaininga signal-to-noise(SN) ratioof
3:1
and a quantification limit (LOQ) of 24.61 ng/mL. The
accuracy and precision of the method was less than 15%
and the correlation coefficient R2 was higher than 0.99.

Fiziologia - Physiology • 2020.30.1(99)Fig. 1. Calibration curve for AAI with the equation of theline (y) and the regression coefficient (R2)
3.2 Analytical performance of the proposed method
by IT-SPME coupled with Cap-LC
After several attempts to find the best combination ofmobile phases, taking into account the properties of AAI,
we concluded that an acetonitrile-water mixture (50% 50%) in the isocratic mode and a pH adjustment using
0.1% formic acid gave the best results in terms of
separation of the analyte and peak intensities from thechromatograms obtained. The flow rate of the tested
mobile phases ranged between 5 and 20 µL/minute, andthe flow rate of 20 µL/minute proved to be suitable for agood intensity and resolution of the chromatographic
peak. To choose the optimal value for analyte detection
we used the Zorbax C18 column specific for hydrophobic
compounds. Another analytical parameter studied, theinjection volume, we started with injections with aminimum volume of 25 µL, then we gradually increased to
50 µL, 100 µL, 500 µL to large volumes of 1 mL to 1.5
mL. Although the highest intensity is directly proportional
to the highest volume, in analytical chemistry there must
be a compromise when it comes to quantities, volumes
and intensities, because even if we apparently get a very
good sensitivity. Therefore, the optimal result was reached
at a volume of 500 mL, as can be seen from the graph in
Figure 2. Making these settings for optimization, AAI was
eluted before 10 minutes.

The IT-SPME coupled with Cap-LC system has many
advantages in terms of analytical performance, being
suitable for environmental samples, such as the samples
in the present study. Using dynamic desorption, theanalyte (AAI) is extracted and preconcentrated inside thecapillary column covered on the inside with a thin film of
extractive phase and then the analyte is transferred to theanalytical column using the mobile phase by switching thevalve position, thus separating and detection of thecompound. In our experiments, we replaced the stainlesssteel injection loop of a conventional injection valve with a
GC capillary column covered with a phase of 35%
dimethylpolysiloxane
65%
diphenylpolysiloxane,
considered suitable for AAI extraction, being ahydrophobic compound.
The final optimal conditions for the detection andquantification of AAI standards were: acetonitrile-water
mixture (with 0.1% formic acid) in a proportion of
50%:50%, injection volume 500 µL, flow rate of 20µL/minute. The addition of only 0.1% formic acid as a pH
modifier significantly increased AAI peaks. The following
image (Figure 3) shows overlapping chromatograms of the
100 ng/mL AAI standard solution, injected in the same
concentration several times, and immediately after each
injection, a clean-up injection was performed directly into
the capillary tube with volumes of water between 50 and
100 µL. This procedure is well known and is applied toincrease the sensitivity and resolution of the analytepeaks. Optimal results were obtained by injecting 50 µL
water immediately after injecting the standard or sample,
observing a proportionality in terms of peak intensity but
also retention time (RT = 6.8 minutes).

Fig. 3. Chromatograms obtained from the same
concentration of 100 ng/mL AAI standard solution withdifferent volumes (0, 50, 100 µL) of clean-up
injections with water

Fig. 2. Bar plots for the volume of injection andthe corresponding area
Fiziologia - Physiology • 2020.30.2(100)

3.3 Comparison of the new method with previously
reported methods
Current approaches for AAs separation detection andquantification include chromatographic methods, mainly
liquid chromatography (LC) coupled with ultraviolet (UV),
fluorescence (FLD) detection or mass spectrometry (MS)[16-23]. Widely, HPLC is considered the routine method95

for detecting AAs and specialized articles identified a
detection limit (LOD) at the nanogram levels [18,19,22,23].
Most HPLC methods focus on AAI and/or AAII
(aristolochic acid II) analysis in various samples, from thesimplest to the most complex. Throughout the process of
optimization of analytical methods, which involves thecomposition and percentage of mobile phases, pH andanalytical columns, the separation and quantification of
AAs is increasingly accurate [18-23,39]. A better analyticalsensitivity for the detection of AAI and AAII in extracts was
obtained with a liquid chromatograph coupled with tandem
mass spectrometry (LC-MS/MS), using an ion trap MS(MS-IT), but the best characteristics Ionizations wereobtained using atmospheric pressure chemical ionization(APCI) and by including ammonium ions in the mobile
phase. Detection limits for AAs are also influenced byinterferences with the sample matrix [40-42].
The toxic contaminants, AAs, have multiple sourcesof entry into the human body: soil - plant - water - food air, therefore they have been declared extremely toxic
compounds by IARC [12]. To support these claims, newimprovements in chromatographic methods in terms of
selectivity and sensitivity have been able to detect AAs at
low concentrations in all these environmental samples,
which could help to understand the entry of AAs into thehuman body.
Studies started by Ivic [8] and continued by Hranjec
et al. [40] demonstrated that Aristolochia clematitis seeds
have an increased content of AAs and deduced that thesephytotoxins have an imperative role in causing BEN.
Afterwards, many research groups have followed thesame direction and developed modern chromatographic

methods to identify and detect AAs in various
environmental samples: extracts from Chinese plants[36,44], soils [18-22,39], groundwater [23] and plants
cultivable in crop fields: wheat and corn [18,22,45]. The
detection of AAs was studied in vegetables grown inlaboratory conditions: peas and cucumbers [16] andlettuce, spring onions and tomatoes [17]. In recent years,
several extraction methods have been proposed usinglarge volumes of solvents.
For the same comparative purpose, we designed thetable below (Table I), which contains a summary ofmodern analytical methods used to extract, detect andquantify AAs from various samples or matrices, but not
only from the Balkan countries but from around theworld. The genus Aristolochia includes up to 600 different
species, present mainly in Asia, and in Romania thecharacteristic species is Aristolochia clematitis . Due tothe ubiquity of this genus of plants, comes the similarity
between the three types of kidney disease with almost
identical pathology: BEN, AAN and CHN. Table 1 focuseson one of the most important analytical parameters, LOD(limit of detection), which is the minimum concentration atwhich the signal can be detected for a specificanalyte. The detection techniques generally used are
chromatographic of various types. The matrices or
samples from which the extraction is made are largely
environmental samples or biological samples. LODs varyfrom 0.02 ng/mL to 0.03 ug/mL. Good sensitivity of 0.02
ng/mL was reached by adding Zn/H+ to induce
nitroreduction, so AAs convert into their respective
aristolactams (AL), which dramatically enhances theiranalytical sensitivity [17-21,33-36,44-53].

Table I. Table comparing various modern methods used for estimating AAI in different types of samples; Principal
characteristics of analytical methods and parameters.
Sample/MatrixExtraction method

Detection technique

LOD

Reference

Environmental
samples(soils/vegetables)

IT-SPME

Cap-LC

8.12
ng/mL

Current
methodMSPE(Solid phase magnetic
extraction)

HPLC

0.05
μg/mL

[46]

Chinese herb soup

Soils from Serbia(Endemic area)

96

Solvent
extraction (methanol:
water: acetic acid = 70:25:5)

LC-MS / MS
(Zn / H)
Transformation
AAI -> AL I (Aristolactam I)

ALI: 62.5
ng/kg

[20]

Fiziologia - Physiology • 2020.30.1(99)Wheat grains, corngrainsand soil(Endemic area)

Extraction solvent (methanol:
water:acetic acid = 70:25:5)

LC-MS / MS
(Zn / H)
AAI -> AL I

MPE (Multiphase Extraction)- Methanol: Water = 60:40

HPLC

2.4 μg/g

Extraction solvent (80%m ethanol 20% aqueous
mixture (90% water
10% formic acid))

HPLC, MS

100
ng/mL

Medicinal plants,
weight loss
supplements

Extraction solvent(m ethanol)

SIA (Sequential Injection
Analysis) andCL (Chemiluminescence)

3 ng/mL

Medicinal herbs

Extraction solvent (70%m ethanol)

SPE (Solid Phase
Extraction) and HPLC

0.03
μg/mL

Wheat grains, corngrains, soils

The extraction solvent(methanol: water: acetic acid
= 70:25:5)

HPLC-FLD and
UPLC - MS / MS

3.3 ng/g

Lettuce leaves
grown in soil
contaminated withAAs (laboratory
conditions)

Extraction solvent (methanol:
water:
acetic acid, 70:25:5 )HPLC-FLD

0.73
ng/mL

[50]

Chinese herbal
medicines

Extraction
solvent ( methanol )LC-FLD

0.271
ng/mL

[51]

Plants, medicinal
plantsSoil and SOM (soilorganic matter)

Herbal foodsupplements
Herbal products,
biological samples(rat serum)
Biological samples(rat urine andplasma)Medicinal herbs

QuEChERS solid phaseextraction method (deionizedwater 2% formic acid,
acetonitrile, buffer salts)
Extraction solvent(m ethanol)

UHPLC-MS

UHPLC-MS / MS

Extraction solvent (Methanol:
Water: Acetic acid = 70: 25:
5)

HPLC-FLD

Extraction solvent (methanol:
water: acetic acid = 70: 25:
5)

LC-FLD

Fiziologia - Physiology • 2020.30.2(100)

AL I:
0.02
ng/mL

[21]

[47]

[19]

[48]

[49]

[18]

1 ng/mL
[52]
0.14
ng/mL

0.09 ng(urine)0.07 ng(plasma)
0.39 ng/ mL[53][ 154 ]

[44]97

4. CONCLUSIONREFERENCES

In this work, IT-SPME coupled to Cap-LC with DAD
detection has been proposed to the determination of AAI
in environmental samples such as soils and vegetables.
First, the experimental conditions were optimized in orderto achieve the proper selectivity and sensitivity. Under the
optimal conditions, the described method offers good
accuracy and reproducibility, lineal answer (R2 > 0.99) and
precision (RSD < 15%) are reached. Finally, highsensitivity is achieved by achieving lower detection limit
can be reached with the processing of large sample
volumes. Some alternative strategies such as increasing
the in-tube capillary length or the size of the loop of thevalve can be applied particularly if lower detection limits
want to be reached. It joins the advantages of both
systems allowing high speed time analysis since
separation is achieved in less than 10 minutes.
The proposed method is cost effective to determine
AAI in environmental samples, has good analyticalcharacteristics and allows the identification andquantification of AAI in appropriate concentrations tocontrol environmental quality. Moreover, the considerable
reduction on the amounts of sample and chemicals inreference to other procedures proposed in the literature.
Compared with previous studies done by other researchgroups, this method is a good alternative.
Although many papers have been published in recent
years, the efficiency of separation needs to be further
improved, especially for the analysis of real evidence.
Most of the proposed methods are used to quantify AAs in
herbal mixtures, which contain relatively highconcentrations of AAs, but may not be sensitive enough todetect low levels of AAs in complex matrices ofenvironmental samples. There is a general trend inanalytical methods towards simplification andminiaturization of the sample preparation process, which
reduces the time of handling and analysis of samples, aswell as the consumption of large volumes of solvents andwaste generation.
This study on chromatographic methods for complex
environmental analysis has a major impact on the healthof certain vulnerable or exposed people, but also on thechemistry of the environment. Further studies would be
useful to clarify the role of soil contamination with otherphytotoxins or environmental factors involved in the
etiology of BEN.

1.

2.

3.

4.

5.

6.

7.
8.
9.

10.
11.

12.

13.
14.

98

Tatu CA, Orem WH, Finkelman RB, Feder GL. The
etiology of Balkan endemic nephropathy: still morequestions than answers. Environ Health Perspect.
1998, 106 (11), 689-700.
Arlt VM, Ferluga D, Stiborova M, Pfohl-Leszkowicz A,
Vukelic M, Ceovic S, et al. Is aristolochic acid a risk
factor for Balkan endemic nephropathy-associatedurothelial cancer? Int J Cancer. 2002;101:500-2.
Voice T, Long D, Radovanovic Z, Atkins J, McElmurry
S, Niagolova ND, et al. Critical evaluation ofenvironmental exposure agents suspected in the
etiology of Balkan endemic nephropathy. Int J Occup
Environ Health. 2006;12:369-76.
Grollman AP, Shibutani S, Moriya M, Miller F, Wu L,
Moll U, Suzuki N, Fernandes A, Rosenquist T,
Medverec Z, Jakovina K, Brdar B, Slade N, Turesky
RJ, Goodenough AK, Rieger R, Vukelic M, Jelakovic
B. 2007. Aristolochic acid and the etiology of endemic(Balkan) nephropathy. Proc Natl Acad Sci USA.
104:12129–12134.
Gluhovschi G, Margineanu F, Velciov S, Gluhovschi
C, Bob F, Petrica L, et al. Fifty years of Balkan
endemic nephropathy in Romania: Some aspects ofthe endemic focus in the Mehedinti county. Clinical
Nephrology, 2011; 75(1): 34-48.
De Broe ME, Chinese herbs nephropathy and Balkan
endemic nephropathy: toward a single entity,
aristolochic acid nephropathy, Kidney Int. 81:6 (2012)
513–515.
Stiborova M, Arlt VM, Schmeiser HH. Balkan endemic
nephropathy: an update on its aetiology. Arch Toxicol.
90 (2016) 2595-615.
Ivic M. Etiology of endemic nephropathy. Lijec Vjesn.
1969;91: 1273-81.
Vanherweghem JL, Depierreux M, Tielemans C, et al.
Rapidly progressive interstitial renal fibrosis in young
women association with slimming regimen including
Chinese herbs, Lancet 341 (1993) 387–391.
Debelle FD, Vanherweghem JL. Nortier J.
Aristolochic acid nephropathy: a worldwide problem,
Kidney Int. 74 (2008) 158–169.
Gillerot G, Jadoul M, Arlt VM, et al. (2001) Aristolochic
acid nephropathy in a Chinese patient: time toabandon the term “Chinese herbs nephropathy”?
American Journal of Kidney Diseases, 38, E26.
IARC Working Group on the Evaluation ofCarcinogenic Risks to Humans. Some traditional
herbal medicines, some mycotoxins, naphthalene
and styrene. IARC Monogr. Eval. Carcinog. Risks
Hum. 82 (2002) 69-128.
Pavlovic NM, Balkan endemic nephropathy - current
status and future perspectives, Clin Kidney J. 6:3(2013) 257–265.
Jadot I, Declèves AE, Nortier J, Caron N. An
Integrated View of Aristolochic Acid Nephropathy:
Update of the Literature. Int. J. Mol. Sci. 2017, 18 (2),297.
Fiziologia - Physiology • 2020.30.1(99)15. Han J, Xian Z, Zhang Y, Liu J, Liang A. Systematic
Overview of Aristolochic Acids: Nephrotoxicity,
Carcinogenicity, and Underlying Mechanisms. Front.
Pharmacol. 2019, 10, 648.
16. Pavlović NM, Maksimović V, Maksimović JD et al.
Possible health impacts of naturally occurring uptakeof aristolochic acids by maize and cucumber roots:
links to the etiology of endemic (Balkan) nephropathy.
Environ Geochem Health. 2013, 35 (2), 215-226.
17. Li W, Hu Q, Chan W. Uptake and Accumulation of
Nephrotoxic and Carcinogenic Aristolochic Acids inFood Crops Grown in Aristolochia clematitis Contaminated Soil and Water, J. Agric. Food Chem.
64: 1, (2015) 107-11.
18. Chan W, Pavlovic NM, Li W et al. Quantitation ofaristolochic acids in corn, wheat grain, and soil
samples collected in Serbia: identifying a novel
exposure pathway in the etiology of Balkan endemic
nephropathy, J. Agric. Food Chem. 64 (2016) 5928–
5934.
19. Gruia AT, Oprean C, Ivan A, et al. Balkan endemic
nephropathy and aristolochic acid I: An investigation
into the role of soil and soil organic matter
contamination, as a potential natural exposure
pathway, Environ Geochem Health 40 (2017) 1437–
1448.
20. Chan CK, Chan KKJ, Pavlović NM, Chan W, Liquid
chromatography-tandem mass spectrometry analysisof aristolochic acids in soil samples collected from
Serbia: Link to Balkan endemic nephropathy. Rapid
Commun Mass Spec. 2019; 34, e8547.
21. Chan CK, Liu Y, Pavlovic NM, Chan W. Developmentof a novel liquid chromatography-tandem mass
spectrometric method for aristolochic acids detection:
Application in food and agricultural soil analyses,
Food Chem. 289 (2019) 673-679.
22. Li W, Chan CK, Liu Y, et al. Aristolochic Acids as
Persistent Soil Pollutants: Determination of Risk for
Human Exposure and Nephropathy from Plant
Uptake, J. Agric. Food Chem. 2018; 66, 1146811476.
23. Tung KK, Chan CK, Zhao Y, Chan KJ, Liu G, Pavlović
NM, Chan W, Occurrence and Environmental Stabilityof Aristolochic Acids in Groundwater Collected from
Serbia: Links to Human Exposure and Balkan
Endemic Nephropathy, Environ. Sci. Technol. 2020;
54, 1554-1561.
24. Li W, Chen Z, Liao Y, Liu H. Separation methods for
toxic components in Traditional Chinese Medicines.
Analytical Sciences. 2005; 21:1019-29.
25. Chan W, Hui KM, Poon WT, Lee KC, Cai Z.
Differentiation of herbs linked to "Chinese herb
nephropathy" from the liquid chromatographic
determination of aristolochic acids. Anal. Chim. Acta.
26. Chan CK, Liu Y, Pavlović NM, Chan W. Etiology ofBalkan endemic nephropathy: an update onaristolochic acids exposure mechanisms. Chem. Res.
Toxicol. 2018;31(11):1109-1110.
27. Campíns-Falcó P, Verdú-Andrés J, Sevillano-Cabeza
A, Molins-Legua C, Herráez-Hernandez R. New
micromethod combining miniaturized matrix solidphase dispersion and in-tube in-valve solid-phase
Fiziologia - Physiology • 2020.30.2(100)

microextraction for estimating polycyclic aromatichydrocarbons in bivalves, J. Chromatogr. A 1211
(2008) 13-21.
28. Muñoz-Ortuño M, Argente-García A, MolinerMartínez Y, Verdú-Andrés J, Herráez-Hernández R,
Picher MT, Campíns-Falcó P. A cost-effective method
for estimating di(2-ethylhexyl)phthalate in coastal
sediments, J. Chromatogr. A 1324 (2014) 57-62.
29. Moliner-Martinez Y, Herráez-Hernández R, VerdúAndrés J, Molins-Legua C, Campíns-Falcó P. Recent
advances of in-tube solid-phase microextraction, 71(2015) 205-213.
30. Li W, Chen Z, Liao Y, Liu H. Separation methods for
toxic components in Traditional Chinese Medicines.
Analytical Sciences. 2005; 21:1019-29.
31. Hashimoto K, Higuchi M, Makino B, Sakakibara I, Kubo
M, Komatsu Y, Maruno M, Okada M. Quantitative
analysis of aristolochic acids, toxic compounds,
contained in some medicinal plants. Journal of
Ethnopharmacology. 1999; 64, 185-189.
32. Cheung TP, Xue C, Leung K, Chan K, Li CG.
Aristolochic acids detected in some raw chinese
medicinal herbs and manufactured herbal products a consequence of inappropriate nomenclature andimprecise labelling Clinical Toxicology. 2006; 44, 371378.
33. Yamasaki K, Tagami T, Kawaguchi M, Okihashi M,
Takatori S, Sakagami Y, Sekita S, Satake M. Simple
and rapid analysis of aristolochic acid contained in
crude drugs and Kampo formulations with solid-phaseextraction and HPLC photodiode-array detection.
Journal of Natural Medicines, 2009; 63, 451 458.
34. Kite GC, Yule MA, Leon C, Simmonds MS. Detecting
aristolochic acids in herbal remedies by liquid
chromatography/serial mass spectrometry. Rapid
Communications in Mass Spectrometry, 2002; 16,585-590.
35. Yuan J, Nie L, Zeng D, Luo X, Tang F, Ding L, Liu Q,
Guo M, Yao S. Simultaneous determination of ninearistolochic acid analogues in medicinal plants andpreparations by high performance liquid
chromatography. Talanta, 2007; 73, 644-650.
36. Trujillo WA, Sorenson WR, La Luzerne P, Austad JW,
Sullivan D. Determination of aristolochic acid inbotanicals and dietary supplements by liquid
chromatography with ultraviolet detection and byliquid chromatography/mass spectrometry: single
laboratory validation confirmation. Journal of AOAC
International, 2006; 89, 942-959.
37. Jou J, Li CP, Schelonka E, Lin C, Wu T. Analysis of theAnalogues of Aristolochic Acid and Aristolactam in the
Plant of Aristolochia Genus by HPLC. Journal of Foodand Drug Analysis. 2004; 12, 40-45.
38. Drăghia LP, Lukinich-Gruia AT, Oprean C, Paunescu
V, Tatu CA. Preliminary study of soil composition fromBalkan endemic nephropathy areas, using a GC-MS
method.
Environmental
EngineeringandManagement Journal, 2019; 18, 2455-2463.
39. Chan CK, Liu Y, Pavlović NM, Chan W, Aristolochic
Acids: Newly Identified Exposure Pathways of thisClass of Environmental and Food-Borne

99

Contaminants and its Potential Link to Chronic Kidney
Diseases, Toxics, 2019; 7, E14.
40. Hranjec T, Kovac A, Kos J, Mao W, Chen JJ,
Grollman AP, et al. Endemic nephropathy: the casefor chronic poisoning by aristolochia. Croatian J Med.
46, 2005; 116-25.
41. Flurer RA, Jones MB, Vela N, et al. Determination ofaristolochic acid in traditional Chinese medicines anddietary supplements J. Lab Inform Bull (of the U.S.
Food and Drug Administration). 2000; 4212: 13.
42. Kite GC, Yule MA, Leon C, Simmonds MS. Detecting
aristolochic acids in herbal remedies by liquid
chromatography/serial mass spectrometry. Rapid
Communications in Mass Spectrometry, 2002; 16,585-590.
43. Stefanovic V, Toncheva D, Atanasova S, Polenakovic
M. Etiology of Balkan endemic nephropathy andassociated urothelial cancer. American Journal of
Nephrology. 2006; 26, 1.
44. Chan W, Lee KC, Liu N, Cai Z. A sensitivity enhanced
high-performanceliquidchromatography
fluorescence method for the detection of nephrotoxic
and carcinogenic aristolochic acid in herbal
medicines. J Chromatogr A. 1164 :1-2 (2007) 113119.
45. Au CK, Zhang J, Chan CK, Li C, Liu G, Pavlovic NM,
et al. Determination of aristolochic acids invegetables:
nephrotoxic
andcarcinogenic
environmental pollutants contaminating a broad
swath of the food supply and driving incidence ofBalkan Endemic Nephropathy, Chem. Res. Toxicol.
2020; 33, 9, 2446–2454.
46. Ji F, Jin R, Luo C et al., Fast determination ofaristolochic acid I (AAI) in traditional Chinese
medicine soup with magnetic solid-phase extraction

100

47.

48.

49.

50.

51.

52.

53.

by high performance liquid chromatography, J.
Chromatogr. A (2019).
Fang L, Tian M, Yan X, Xiao W, Row KH. Dual ionicliquid-immobilized silicas for multi-phase extraction ofaristolochic acid from plants and herbal medicines, J.
Chromatogr. A 1592 (2019) 31-37.
Oraby HF, Alarfaj NA, El-Tohamy MF. Goldnanoparticle-enhanced
luminol/ferricyanide
chemiluminescence system for aristolochic acid-I
detection in medicinal plants and slimming products,
Green Chem Lett Rev 10:3 (2017) 138-147.
Ge Y, Guo P, Xu X, et al. Selective analysis ofaristolochic acid I in herbal medicines by dummy
molecularly imprinted solid-phase extraction and
HPLC, J. Sep. Sci. 40 (2017) 2791–2799.
Wang Y, Chan W. Determination of aristolochic acids
by high-performance liquid chromatography withfluorescence detection, J. Agric. Food Chem. 62(2014) 5859-5864.
Vaclavik L, Krynitsky AJ, Rader JI. Quantification ofaristolochic acids I and II in herbal dietary
supplements by ultra-high-performance liquid
chromatography–multistage fragmentation mass
spectrometry, Food Addit Contam Part A, 31 (2014)784-791.
Kuo CH, Lee CW, Lin SC et al. Rapid determinationof aristolochic acids I and II in herbal products andbiological samples by ultra-high-pressure liquid
chromatography-tandem
mass
spectrometry,
Talanta. 80 (2010) 1672–1680.
Yue H, Chan W, Guo L, Cai Z. Determination ofaristolochic acid I in rat urine and plasma by highperformanceliquidchromatography
withfluorescence detection, J Chromatogr B. 877:10(2009) 995-999.

Fiziologia - Physiology • 2020.30.1(99)DEZVOLTAREA UNEI METODE MINIATURIZATEPENTRU ESTIMAREA ACIDULUI ARISTOLOHIC I(AAI) ÎN PROBE DE MEDIUREZUMAT
Acidul Aristolochic I (AAI) este cunoscut ca principalul compus nitrofenantrenic otrăvitor aparținând grupului mare de aciziaristolochici (AA), produs în mod natural de genul Aristolochia și Asarum. Această fitotoxină este considerată principalul
factor cauzal implicat în nefropatia endemică balcanică (BEN), o boală cronică renală cronică indusă de mediu, prezentă
în anumite zone balcanice, inclusiv în câteva sate din România. În acest articol, am evidențiat diferite tipuri de tehnici deextracție utilizate pentru detecția și cuantificarea AAI în diferite probe de mediu, dezvoltate pentru metode specifice decromatografie. Pentru a extinde sfera studiilor noastre anterioare privind apariția și distribuția contaminării cu AAI în
sistemele „sol-plantă-mediu”, am propus o nouă metodă sensibilă a tehnicilor de extracție miniaturizate: microextracția înfază solidă în tub (IT-SPME) cuplat cu cromatografie lichidă capilară (Cap-LC). Separarea cromatografică și cuantificarea
AAI au fost efectuate în condiții izocratice, folosind o coloană LC sub formă de particule (Zorbax SB-C18) și DAD conectate
la coloana capilară (TRB-35) prin intermediul unei supape de comutare. Liniaritatea a variat între 10–100 ng/mL, atingândo limită de detecție (LOD) de 8.12 ng/mL.
Metoda propusă este economică pentru determinarea AAI în probele de mediu, are caracteristici analitice bune și permite
identificarea și cuantificarea AAI în concentrații adecvate pentru a controla calitatea mediului. Caracterizarea probelor poatefi realizată în mai puțin de 10 minute.
Cuvinte cheie: acid Aristolochic I; Nefropatie endemică balcanică; Eșantioane de mediu; Extracţie; Cromatografie lichidă

Fiziologia - Physiology • 2020.30.2(100)101