FIZIOLOGIA
physiology
FOUNDING EDITORCHIEF EDITOR
CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERCARMEN PANAITESCUCARMEN TATUFLORINA BOJINMIHAI NECHIFORSORIN RIGAGABRIELA TANASIEDACIANA NISTORMARIUS GEORGESCUEDITORIAL BOARD
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

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Fiziologia - Physiology • 2019.29.1(97)CONTENTS
In memoriam: Docent Professor Dr. Gheorghe Arsenescu ........................................................................... 4
Prof. Dr. Marius Sabau

Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes for Cardiac Applications .................... 5
Barbulescu GI, Bojin F, Ordodi V, Anghel S, Gavriliuc O, Paunescu V
CAR-NK Cell-Based Therapy: An Era of a New Potential Immunotherapy ................................................ 13
Szekely FAE, Zogorean R, Anghel S, Paunescu V
Neurodegenration and Neuroprotection in Severe Retinal Dysfunction ................................................... 22
Istrate BM, Vilciu C
Tuberculous Pleural Effusion or Dextroposition of the Heart? A Case Report ......................................... 26
Nicolosu D, Georgescu M, Cioboata R
The Role of Vitamin D in the Pathogenesis of Multiple Sclerosis .............................................................. 30
Albu VC, Sandu RE, Bumbea AM, Tartea AE, Bogdan C, Florescu AO, Burada E, Tartea GC,
Malin R, Barbulescu AL

CUPRINSIn memoriam: Profesor Docent Dr. Gheorghe Arsenescu ............................................................................. 4
Prof. Dr. Marius Sabau

Aplicații clinice pentru cardiomiocite obținute din celule stem pluripotente induse umane .................... 5
Barbulescu GI, Bojin F, Ordodi V, Anghel S, Gavriliuc O, Paunescu V
Terapia cu celule CAR-NK: era unei noi potențiale imunoterapii ............................................................... 13
Szekely FAE, Zogorean R, Anghel S, Paunescu V
Neurodegenerarea și neuroprotecția în disfuncțiile severe ale retinei ...................................................... 22
Istrate BM, Vilciu CRevărsat pleural tuberculos sau dextropoziția inimii? Raport de caz ....................................................... 26
Nicolosu D, Georgescu M, Cioboata R
Rolul vitaminei D în patogenia sclerozei multiple ........................................................................................ 30
Albu VC, Sandu RE, Bumbea AM, Tartea AE, Bogdan C, Florescu AO, Burada E, Tartea GC,
Malin R, Barbulescu AL

Fiziologia - Physiology • 2019.29.1(97)3In memoriam
Docent Professor Dr. Gheorghe Arsenescu
1919-1987
July 15th of this year, 2019, marks the 100th
anniversary of the birth of Professor Gheorghe Arsenescu,
he who headed the department of Physiology at our
University for almost a quarter of a century, between 1963
and 1987.
75 years previous, in September 1944, a young
medical officer, a recent graduate of the Military-Sanitary
Institute of Bucharest, originally from Cocu-Arges, foughtwith weapon in hand, after all the officers of his subunit
were killed, to conquer back the Sângeorz hill near Oarba
de Mureș. Many of the 11,000 Romanian soldiers whodied in this battle, one of the fiercest fought by the
Romanian army for the liberation of northern
Transylvania, under Hungarian occupation following the1940 Vienna Dictate, were killed under his very own eyes.
For these deeds, doctor Gheorghe Arsenescu wasawarded with the order of Mihai Viteazul with swords, a
medal only ever bestowed to a few, and those few only
ever officers distinguished in combat.
The city of Tg. Mureș was liberated by Romanian andSoviet troops on the 28th of September, 1944, while thefighting in Oarba was still raging, to end only on the 6th ofOctober.
Destiny has contrived to make his last and mostfruitful years be spent here in Tg.Mureș, not far from theplace where he risked his life so that we can be who we aretoday.
After the war, Gheorghe Arsenescu became a scientist
at the Institute of Normal and Pathological Physiology"Daniel Danielopolu" in Bucharest. He returned to Tg.
Mureș 19 years later, in 1963, as Associate Professor andthen Professor at the Department of Physiology, a position
which he held until his death in 1987.
As a teacher, he was always preoccupied with the levelof preparation of his students, whom he treated withunrivalled understanding and kindness. His lectures wereof the highest level, perhaps harder to understand bysome students, but admired and respected by all for theunequalled way in which they were presented.
Both leader and mentor to his doctoral students,
among whose number I am honored to count myself, heendeavored to make of them his true friends andcollaborators, to teach them to be exigent with themselves,to spare no effort in achieving something worthy of the4trust he had granted them. He was sparing with praise,
but deep down in his heart he always knew how to holdthose he loved and cherished in the high place they
deserved.
His greatest passion was research, a passion learned
from his mentor Professor Danielopolu.
In the 24 years spent at Tg. Mures he was everpreoccupied with everything novel, and together with theteam he led he created new fields of activity. He introduced,
for the first time in our country, experimental myocardial
microelectrophysiology, the use of radioisotopes inexperimental cardiology, clinical use of orthogonalelectrocardiography and vectorcardiography, as well as
studies on the automatic computer processing of ECG
data.
His more than 150 scientific works made him wellknown, in the country and abroad. In his capacity as amember of the International Council of the Society ofElectrocardiology, he participated with presented papers
at 15 international congresses of the society.
He achieved everything through hard, often
exhausting, work. His was the legacy of a long line ofpeople who knew they had to strive forward with nothing
beyond their own wits and strength of arms.
He was a modest, honest and fair man, withtremendous will and strength, with a robustness you
would not have guessed in his ascetic body.
He was passionate about history especially that of his
country of which he was supremely proud, like many in his
area of the Arges sub-Carpathians. A colonel Gheorghe
Arsenescu is known today as an anti-communist
resistance fighter in the Muscel area, who was executedfor his struggles in 1962.
Even as the merciless illness laid him dying on his
hospital bed, he continued to work on the manuscript ofa lecture, until his final moments. He died in June 1987
and was buried in his hometown, with his forefathers, inthe land he had loved so deeply.
Now, 100 years after his birth, we remember our
teacher, with gratitude for all that he has done so that we
who followed him can take his faith further and farther, to
new heights.
Prof. dr. Marius Sabău
UMF Targu Mures
Fiziologia - Physiology • 2019.29.1(97)HUMAN INDUCED PLURIPOTENT STEM CELLS-DERIVED
CARDIOMYOCYTES FOR CARDIAC APPLICATIONSBĂRBULESCU GRETA IONELA, BOJIN FLORINA, ORDODI VALENTIN,ANGHEL SIMONA, GAVRILIUC OANA, PĂUNESCU VIRGIL
Centre for Gene and Cellular Therapies in Cancer - OncoGen, Clinical County Emergency Hospital Timişoara, Romania“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT
Induced pluripotent stem cells (iPSCs) are a type of cells that can be generated directly from adult cells and can bedifferentiated into many cell lines, including cardiomyocytes. The first reprogramming methods had low efficiency but lately
safe lines of iPSCs without insertional mutations have been achieved. The human iPSCs technology offers a unique
opportunity to obtain patient-specific hiPSC-derived cardiomyocytes. This platform allows iPSCs to be used for drug testing,disease modeling and cell therapy development. In this review, we discuss the progress and applications regarding human
iPSCs, including their potential, limitations and future directions.
Keywords: cardiomyocytes, embryonic stem cells, end-stage heart failure, induced pluripotent stem cells, long QT
syndrome, torsade-de-pointes.

1. INDUCED PLURIPOTENT STEM CELLS –FROM PAST TO FUTURE
The discovery that somatic cells could be used to obtainpluripotent stem cells (PSCs) has made revolutionarychanges in stem cell research. Induced pluripotent stem
cells (iPSCs) from mouse embryonic and adult fibroblast
cultures were first reported in 2006 by Yamanaka and
Takahashi [1]. After that, the same researchers obtained
iPSCs from human fibroblasts by viral overexpression of thesame specific transcription factors with mouse iPSCs,
OCT3/4, SOX2, Klf4 and c – Myc. The obtained human
iPSCs were very similar to human embryonic stem (ES) cells
in many aspects. Regarding morphology, the iPSCs hadlarge nuclei and scant cytoplasm, like ES cells. They
showed feeder dependency with no attachment to gelatincoated tissue-culture plates and similar proliferation as ES
cells. iPSCs expressed human ES cell-specific surface
markers like stage-specific embryonic antigen (SSEA-3,
SSEA-4), tumor-related antigen (TRA-1-60, TRA-1-81) and
NANOG protein. The studied cells expressed
undifferentiated ES cell-marker genes such as: OCT3/4,
SOX2, NANOG, growth and differentiation factor 3 (GDF3),
fibroblast growth factor 4 (FGF4), reduced expression 1(REX1) and many others. IPSCs showed high telomerase
activity and teratoma formation when injectedsubcutaneously to immunodeficient mice. Also, human
iPSCs showed in vitro differentiation to multiple cell lines likeneuronal or cardiac [2]. After this important discovery, iPSCs
were obtained from many other cell-specific tissues, such as
keratinocytes or blood cells [3,4].
Initially, retroviruses and lentiviruses were used to obtain
iPSCs from somatic human cells. The main risk using thesetechniques was the development of insertional mutations in
the cells. To avoid it, other methods of reprogramming withnonintegrating viruses were studied. A good expression
vehicle for generating human iPSCs was adenovirus being a
non-integrating virus. The disadvantage of this method washaving a low efficiency [5]. A much safer and efficient methodof obtaining human iPSC was using non-viral reprogramming
methods like mRNA transfection [6], removable PiggyBac
transposons [7] and plasmid vectors [8].
The low efficiency of the reprogramming methods was
significantly improved by adding different chemicalcompounds such as valproic acid [9], sodium butyrate [10]or creating a hypoxic environment [11]. From these, valproic
acid and sodium butyrate are the most commonly used in
obtaining human iPSCs.

Received February 16th 2019. Accepted June 20th 2019. Address for correspondence: Barbulescu Greta, PhD student, ”Victor Babes”
University of Medicine and Pharmacy Timisoara, Department of Functional Sciences, Immunology Discipline, Eftimie Murgu Square No. 2A,
RO-300041, Timisoara, Romania; Clinical Emergency County Hospital “Pius Brînzeu” Timișoara, Centre for Cellular and Gene Therapies inCancer – OncoGen, Liviu Rebreanu Blvd. 156, RO-300736 Timişoara, Romania; phone: +40733177583; e-mail: greta.barbulescu@gmail.com

Fiziologia - Physiology • 2019.29.1(97)5Human iPSCs give patient-specific derived cells being afaithful genetic copy of the cells taken from a donor. Theiruse comes with its own challenges and limitations such asdifficulty in maintaining fully differentiated cardiomyocytes(CMs) in culture. Reprogramming human adult somatic cells
into iPSCs solved the ethical issues of using human ES cells
in research.

2. CHARACTERIZATION OF INDUCEDPLURIPOTENT STEM CELLS LINES
Since more than a decade ago, when iPSCs have been
discovered, a great progress was made in stem cell biology
and regenerative medicine. This progress came together
with an important concern for their safety and use in clinicalapplications and disease modeling. Unfortunately, studies
showed that iPSCs could sometimes develop genetic
abnormalities during reprogramming or cell culture, being
unqualified for further use. Researchers advise the genomeof iPSCs to be monitored regularly during experiments,
otherwise the tumorigenic risk of such cells could increase.
One factor that can produce genomic alterations is a
prolonged cell culture. The first reported abnormality in iPSC
lines was aneuploidy [12]. In the same time, it is very
important that culture conditions to not influence the cell
genetic integrity. Jacobs et al. showed in a study that higherdensity culture resulted in DNA damage and genome
instability [13]. Another study demonstrated that enzymatic
single-cell passaging can produce karyotype abnormalities
to human iPSCs genome such as trisomy 12 or 20q11.21
amplification [14].
Studies showed that the process of reprogramming cancause genomic alterations when speaking about methods
using integrating vectors, such as human retroviruses. To
overcome this problem, other methods were preferred suchas plasmids or Sendai viruses. Variable levels of aberrant
DNA methylation were described in some studies as
epigenetic differences between iPSC lines. Differences in
DNA methylation was found especially in iPSCs derived
from different sources such as CD34+ cells from peripheral
blood or adult dermal fibroblasts [15]. Another study had
controversial results showing that genomic patterns ofdifferent iPSC lines were similar from different cell sources
when using the same reprogramming approach [16]. These
problems can be partially resolved using different methods
to decrease the genomic alterations during cell
reprogramming such as supplementation of the medium
with nucleosides [17]. However, some studies concluded
that these changes in the iPSCs genome during the
reprogramming protocol are not so harmful and usually don’t
stop the usage of the cells for research [18].
A new way of genome editing called clustered regularlyinterspaced short palindromic repeat (CRISPR)-Cas systems
provide adaptive immunity against genomic alterations. Thisis a simple platform to generate site-specific nucleases (SSN)
6

for targeting genome mutations. It also passes this resistancemechanism vertically to its progeny [19].
Initially, human iPSCs were cultured on mouseembryonic fibroblasts (MEFs) feeder cell lines. This protocol
was efficient and fast but could come with the disadvantageof transferring animal pathogens and inducing immune
response. Human dermal fibroblasts (HDFs) were also used
but they could allow contamination which can be a problem
when using the cells in therapeutic approaches. The solution
for these problems is to replace the MEFs and HDFs feeder
cell lines with something else. For human iPSCs, the field ismoving towards feeder free culturing on Matrigel insupplemented media. Xeno-free media makes things moreclinical applicable [20].

3. CARDIOMYOCYTES DERIVED FROMHUMAN iPSCs
First attempt in obtaining cardiomyocytes from human
iPSCs was by using embryoid bodies but with a poor
efficiency, limiting their utility for further applications [21].
The use of monolayer culture methods has been developed
as a solution for a better cardiomyocyte production.
Exposing the human PSCs to activin A and bonemorphogenetic protein 4 (BMP4) in RPMI/B-27 medium wasreported to be more efficient generating around 30%
cardiomyocytes in the H7 human ESC line [22]. A protocol
for obtaining cardiomyocytes upgraded the previous one byusing TGF-β super family growth factors, lacking insulin in
the differentiation medium and using GSK3 (glycogensynthase kinase 3) inhibitor pretreatment of undifferentiatedhuman PSCs with a result of 30-90% cardiomyocytes acrosshuman PSC lines. Regulation of Wnt/ β-catenin using small
molecules or shRNA showed to be a reliable method for
obtaining cardiomyocytes from human iPSCs in amonolayer culture. Using this technique, cardiomyocytes
showed a high degree of purity (85%) and matureelectrophysiological properties [23].
When human iPSCs are differentiated into
cardiomyocytes they become a heterogeneous mixture ofseveral subtypes, including ventricular cardiomyocytes(VMs), atrial cardiomyocytes (AMs) and CMs from thesino-atrial node (SAN). So, the idea was to create each
CM subtype using physiological and molecular markers to
help discriminate them. A study conducted by Blazeski
showed that most of the protocols are generating
ventricular-like cells [24]. Protocols for AMs differentiation
are not that efficient than those for VMs. Another study
showed that relatively homogeneous VMs and AMs
populations can be derived from human ESCs by
regulating Noggin and retinoid signals [25]. CMs from thesino-atrial node are still difficult to produce. One of themost efficient protocols of obtaining SAN cells is by
inhibition of neregulin1β/ErbB signaling and upregulationof SAN-CMs specific genes [26].
Fiziologia - Physiology • 2019.29.1(97)The main problem when obtaining CMs from iPSCs istheir immaturity and the fact that they share more similarities
with fetal than with adult human cardiomyocytes. Early
hPSC-CMs are mostly round and small (like fetal
cardiomyocytes), becoming later more elongated but not asmuch as adult cardiomyocytes [27]. There are fourcharacteristics used to prove cardiomyocyte’s maturity:
composition and organization of the contractile apparatus,
specific
genes
expression,
metabolism
andelectrophysiological features. Studies showed that
membrane composition, specific intracellular structures(such as the T-tubules which give the maturity of CMs arelacking in the in vitro adult cardiomyocytes), gap junctions
are different in natal CMs when compared with CMs
obtained from iPSCs. All of these can slow the conduction
leading to arrhythmias [28]. When analyzing the specificgene expression (such as RyR2, SERCA, NCX, Hey2, Mlc2v,
etc.), one can see that they are present in both CMs – nativeand in vitro with some differences in their expression levels[29]. Regarding the metabolic changes during CM
maturation, embryonic glycolysis is changed to adult fatty
acid β-oxidation. Also, a high oxidative redox potential isrequired in mature CMs [30]. Maybe the most importantcriteria when analyzing CM maturation is its function. For
example, the expression ratio of β1/ β2 adrenergic receptors
seems to be extremely important [31]. Human PSC-CMs
have a small contraction force. Because of insufficient Ca2+
release and uptake, in human PSC-CMs there is a negative
force-frequency relationship [32].
As explained above, it is very important that iPSC-CMs
to be very similar to native CMs so that can be used for
disease modeling and cardiac regeneration.

4. PREDICTION OF DRUG – INDUCED ARRYTHMIA
Before approving drugs for use, the potential to produce
Torsade-de-Pointes (TdP) is tested by evaluating their ability
to block the human ether-ά-go-go related gene (hERG)
channel in vitro and assessment for QT prolongation risk invivo according to the ICH S7A/B guidelines. TdP is apolymorphic ventricular tachycardia that can lead to sudden
cardiac death when it’s not treated immediately.
Latest studies in the field tried to evaluate drug-induced
cardiotoxicity using human induced pluripotent stem cellderived cardiomyocytes. This method may offer a better
evaluation of cardiac safety and efficacy for tested drugs.
One important example in this direction is theComprehensive in Vitro Proarrhythmic Assay (CiPA)
initiative to detect drug-induced Torsade-de-Pointes (TdP).
The main purpose of this initiative is to categorize drugs into
high, intermediate and low risk of TdP. Human iPSCs areused to confirm in silico reconstructions of theelectrophysiologic effects of drugs. Changes in theextracellular field potentials of active human SC-CMs using
multielectrode array (MEA) platforms are observed as a
Fiziologia - Physiology • 2019.29.1(97)result from drugs interaction [33]. One study published in2015 has demonstrated the ability of hSC-CMs to detect
delayed repolarization due to drugs exposure and holds
promise to a new method to predict arrhythmogenic
potentials [34]. There are still limitations when using human
SC-CMs in drug safety testing because of theirelectrophysiological immaturity. Efforts are made in thisdirection to create more mature CMs from stem cells.
Another attempt to use human iPSC-CMs in predictionof drug-induced arrhythmia came from Japan Cardiac
Safety Assessment (JiCSA). They studied the torsadogenic
potential of 60 drugs and compare it with CiPA initiative, theresults being extremely similar [35].
In the last years, a lot of studies have proposed a new
drug induced torsadogenic risk system using human iPSCCMs and the MEA system. Most of them compared theresults with CredibleMeds®. The conclusion was that using
human iPSC-CMs is reliable and has the potential to replace
the hERG assay for torsadogenic risk prediction [36].

5. iPSCs IN CARDIO – ONCOLOGY
Cardiotoxic effects from cancer therapy using
chemotherapeutic agents, including anthracyclines,
antimetabolites, alkylating agents, tyrosine kinase inhibitors(TKIs), proteasome inhibitors (PIs) are a major cause ofmorbidity. It was necessary to develop preclinical models to
predict human cardiotoxicity. The underlying mechanism of
cardiotoxicity due to chemotherapeutic agents have notbeen fully elucidated until now. The most frequently usedagents have the following cardiovascular effects: decline inleft ventricle ejection fraction (LVEF), ischemia,
cardiomyopathy,
hypertension,
vascularevents,arrhythmias, myocarditis, etc.
Human iPSC-CMs have been used in cardiovascularresearch exploring the cardiotoxicity of different
chemotherapeutic agents being a reliable alternative toinvasive biopsy of human cardiac tissue. The most studied
drug until now is doxorubicin, which belongs to anthracycline
family. Doxorubicin inhibits the function of topoisomerase 2B(TOP 2B) causing cell death of cardiomyocytes.
Cardiotoxicity of doxorubicin occurs in a dose-dependent
manner [37]. A study published by Burridge et al. studied thedifferent response to doxorubicin in human iPSC-CMs
derived from healthy controls, patients treated withdoxorubicin without cardiotoxicity (DOX) and patients withclinical cardiotoxicity due to doxorubicin (DOXTOX). The
DOXTOX cells showed reduced cell viability, arrhythmic
beating, and sarcomere disarray. Using human iPSC-CMs
can unravel the molecular mechanisms of interindividual
variation in toxicity induced by doxorubicin. It also holds
promise for discovery of new cardio protectants such as NAcetyl Cysteine (NAC) [38]. When comparing the effects ofdoxorubicin on 2D and 3D models derived from human
iPSC-CMs, different cardiotoxic responses have been
7

shown, with an increased resistance in the 3D model [39].
Using human iPSC-CMs to create devices such “organ-ona-chip” provides an opportunity to study cardiotoxicity ofchemotherapeutics. In a study published by Zhang et al.
doxorubicin induced cardiotoxicity, increased CK-MB levels
and arrhythmias [40].
Monoclonal antibodies have revolutionized the treatmentof cancer. Unfortunately, they also come with side effects,
including cardiotoxicity. For example, trastuzumab promotes
oxidative stress and downregulation of TOP2b gene
expression in cardiomyocytes [41]. A recent study using
human iPSC-CMs showed that trastuzumab is blocking the
ErbB2/4 pathway that offers cardioprotection [42].
Using iPSC-CMs in chemotherapeutics testing maychange the way drugs are screened, becoming a road topersonalized medicine.

6. iPSCs AS A PLATFORM FOR CARDIACDISEASE MODELING
Human iPSCs create patient-specific iPSC-derived
cardiomyocytes in vitro as a platform for cardiac diseasemodeling. In this way, a lot of hereditary cardiac conditions
like arrhythmias and cardiomyopathies can be studied.
6.1. Channelopathies
A lot of recent studies used iPSCs for exploring thefundamental mechanisms underlying proarrhythmic diseasephenotypes like long QT syndrome, short QT syndrome,
Brugada syndrome and catecholaminergic polymorphic
ventricular tachycardia (CPVT). The maturity of iPSC-CMs
is very important here so the molecular and structuralcharacteristics, action potentials (APs), membrane currents
and excitation-contraction coupling must be examined.
Long QT (LQT) syndrome is a life-threatening inherited
disease characterized by a prolonged ventricularrepolarization. There are three main subtypes of LQT
syndrome: LQT1, LQT2 and LQT3 [43]. LQT1 syndrome is
the most frequent and it’s due to mutations in the KCNQ1
gene, encoding the α subunit of the K+ channel responsibleof IKs. The first study on LQT1 using human iPSC-CMs was
published by Moretti et al. Action potentials of human iPSCCMs were significantly prolonged in LQT1 probes compared
to controls [44]. β-blocker (propranolol) was tested on iPSCCMs from LQT syndrome patients showing beneficial effects
on AP duration [45].
Described above, the combination of CRISPR/Cas9 withinduced pluripotent stem cell technology can develop thestudy of hereditary cardiac arrhythmias. A study publishedby Makita et al showed that mutations in calmodulin genes
caused early onset severe LQTS [46]. The mutations can becorrected by allele-specific knockdown using CRISPR
technology, providing insights into new therapies [47].

8

Patient specific human iPSCs were also used toinvestigate CPVT. This arrhythmia is mainly caused bymutations in RYR2 (ryanodine receptor) or CASQ2(calsequestrin). Human iPSC-CMs with these mutations
showed an increased concentration of intracellular calcium
and increased elementary Ca2+ release events after the
addition of β adrenoreceptor agonist. Dantrolene was shown
to ameliorate the disease, suppressing stress-induced
arrhythmic events in CPVT human iPSC-CMs [48]. Besides
dantrolene, carvedilol and flecainide were shown to haveantiarrhythmic effects in cardiomyocytes derived from CPVT
patients [49].
6.2. Cardiomyopathies
Besides cardiac arrhythmias, human iPSC-CMs havebeen used to study cardiomyopathies in vitro.
Familial dilated cardiomyopathy (DCM) is a disordercharacterized by ventricular dilatation, systolic dysfunction,
progressive heart failure and possibly development ofarrhythmias. In a study published by Sun et al, theresearchers evaluated the TNNT2 mutation (p.R173 W) in
human iPSC-CMs from patients with familial DCM. This
gene is expressed specially in the heart and regulates thecontraction of cardiomyocytes. Comparing CMs from iPSCs
carrying TNNT2 mutation with CMs from iPSCs of healthy
controls, the first category exhibited an altered myofilament
organization, altered Ca2+ handling and decreased
contraction force. Some treatments for improving
sarcomeric organization of CMs from patients with familial
DCM were tested, showing that β-blockers (metoprolol)
were protective [50].
Hypertrophic cardiomyopathy (HCM) is associated withabnormal thickening of the left ventricular myocardium,
being one of the most common hereditary cardiac diseases.
There are still no specific HCM therapies making thisdisease an attractive target for in vitro disease modeling.
The pathogenic mechanism underlying the development of
HCM was evaluated in a study published by Han L et al.
Human iPSC-CMs from a patient with HCM caused by the
MYH7 mutation were reported. Structural and functional
abnormalities like increased intracellular calcium
concentration and increased expression of HCM related
genes and proteins were found in these cells. Calcium
channel antagonists were proven to be benefic for human
iPSC-CMs from HCM patients [51].
This finding indicates the potential of human iPSC-CMs
for valid disease models and patient-specific therapies.

7. CARDIAC CELL THERAPY
Treatment efficiency in heart failure is limited for patientswith severely decreased cardiac function. Therefore, cardiac
cell therapy with human iPSC-CMs is a promising strategy
for patients with end-stage heart failure. Transplanted
Fiziologia - Physiology • 2019.29.1(97)cardiomyocytes derived from pluripotent stem cells arehomozygosity. The grafted cardiomyocytes showed
expected to improve cardiac function.
electrical coupling with the host cardiomyocytes and no
A lot of studies have demonstrated the efficacy ofevidence of immune rejection. The results are promising but
cardiac stem cell therapy in small animal model. A study
further research to evaluate post-transplant arrhythmias is
published by Rojas et al analyzed the engraftment andneeded [56].
functional effects of iPSC-CMs in a murine model of
The first clinical transplantation of human ESC-cardiac
myocardial infarction. iPSC-CMs were transplanted into theprogenitor cells embedded into a fibrin scaffold wasmice hearts 14 days after left anterior descending artery
performed to a 68 years old female patient, diabetic and with(LAD) ligation. Results showed that transplanted iPSC-CMs
NYHA (New York Heart Association) III heart failure despite
formed mature grafts within the myocardium, also
maximal medical therapy due to an extensive antero-lateralsignificantly improved myocardial remodeling and pumpinfarction. Three months after the surgery, thefunction 28 days after LAD-ligation [52].
symptomatology improved, LVEF increased with 10% and a
Recent studies have shifted towards large animal
new-onset contractility was evident at echocardiography. No
models. In a study conducted by Chong et al, one billion
complications such as teratoma formation or arrhythmias
human embryonic stem cells-derived cardiomyocytes (ESCwere reported [57].
CMs) were injected into the heart of adult pigtail macaques
Cardiac myocytes derived from human ESCs are very
two weeks after induced myocardial infarction. Using asimilar to those derived from iPSCs so future approaches
multi-drug regimen containing cyclosporine, methyl
using iPSC-CMs for cardiac therapy should be studied.
prednisone and basiliximab attenuated the immune rejection
and improved survival of human ESC-CMs.
However, transplanted human ESC-CMs did notsurvive at 140 days after transplantation so
remuscularization as a mechanism for the betterrecovery of left ventricular function observed was
excluded. A limitation of the study was therelatively small number of animals (32 monkeys)[53]. Extrapolating the experiment to the humanheart, which is ten times bigger than a macaque’s
heart, an unrealistic number of human ESC-CMs
would be needed to be injected into the humanmyocardium.
To overcome the poor survival of injected cells
into the myocardium, patch-form cardiomyocytes
have been tested in different studies. Engineered
heart muscle from human ESC-CMs
Fig.1. Application of human induced pluripotent stem cell-derived cardiomyocytes
transplantation in a chronic myocardial infarction(iPSC-CMs) in drug discovery, disease modeling and cardiac cell therapy
immunosuppressed rodent model led to highengraftment rates with no significant cell loss andprogressive maturation of human cardiomyocytes. Also, no
teratoma formation was observed. However, ESC-CMs
CONCLUSIONS
engraftment could not be correlated with the functional
Patient-derived iPSC-CMs provide a reliable model tocardiac improvement in the mice [54].
study human heart tissue. They have been shown to play an
Lei Ye et al. reported engraftment of human iPSCimportant role in preclinical toxicity testing of drugs, disease
derived cardiomyocytes, endothelial cells and smooth cells
modeling and brings hope for transplantation therapies in
into a porcine model of myocardial infarction. The 3D fibrin
patients with end stage heart failure. iPSCs are a powerfulpatch loaded with insulin growth factor (IGF) improved left
tool that can provide a unique insight into therapeutic
ventricular function, reducing the infarct size, without
approaches for cardiac inherited disease management.
inducing ventricular arrhythmias [55]. Anyway, researchers
Using novel techniques like CRISPR-Cas9 to geneticallyconcluded that studies with longer follow-up periods are
modify iPSCs will allow a high number of mutations to beneeded to ensure the benefits or potential side effects of thestudied in the next years. Despite their promising use in thetreatment.
future, there are still some obstacles that need to be
The latest studies are focused on allogeneic
overcome like improving maturity of human iPSC-CMs.
transplantation models using iPSC-CMs, providing a major
The future aims to create CMs in situ for heart
benefit in terms of immune rejection. Shiba et al performed
regenerative therapy and with all the progress in the lastan allogeneic transplantation experiment with iPSC-CMs
years, this future may not be that far.
from monkeys with major histocompatibility complex
Fiziologia - Physiology • 2019.29.1(97)9Acknowledgement: This work was supported by the grant“Development of BIOnanotechnologies based on Extracellular
Vesicles, applied in early diagnostic, prognosis and therapy ofatherosclerotic disease (BIOVEA)”, Project code: PN-III-P1-1.2PCCDI-2017-0527, Project number 83PCCDI/2018.

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11

APLICAȚII CLINICE PENTRU CARDIOMIOCITE
OBȚINUTE DIN CELULE STEM PLURIPOTENTEINDUSE UMANE
REZUMAT
Celulele stem pluripotente induse (iPSCs) sunt un tip de celule ce pot fi generate direct din celule adulte și se pot diferenția
spre mai multe linii celulare, inclusiv spre linia cardiomiocitara. Primele încercări de reprogramare au avut eficiență scăzută,pentru ca mai apoi să se obțină linii sigure, fără mutații de iPSCs. Această tehnologie oferă o oportunitate unică de a obține
cardiomiocite pacient-specifice derivate din iPSCs. Această platformă permite utilizarea iPSCs pentru testarea
medicamentelor, investigarea afectiunilor cardiace ereditare cât și pentru terapiile regenerative. Vom discuta în acest review
progresul si posibilele aplicații ale celulelor stem pluripotente induse umane, inclusiv potențialul și limitările acestora.
Cuvinte cheie: cardiomiocite, celule stem embrionare, insuficiență cardiacă stadiu final, celule stem pluripotente induse,sindrom QT lung, torsada vârfurilor.

12

Fiziologia - Physiology • 2019.29.1(97)CAR-NK CELL-BASED THERAPY: AN ERA
OF A NEW POTENTIAL IMMUNOTHERAPYSZEKELY FLAVIA ANNE-ELISE1,2, ZOGOREAN ROXANA1, ANGHEL SIMONA1,3,PĂUNESCU VIRGIL1,3
1Clinical Emergency County Hospital “Pius Brînzeu” Timișoara, Centre for Cellular and Gene Therapies in the Treatment of Cancer – OncoGen
2Emergency Hospital for Children Louis Țurcanu Timișoara
3”Victor Babes” University of Medicine and Pharmacy Timisoara, Department of Functional Sciences, Immunology Discipline

ABSTRACT
Immuno-oncology field is in a continuous process of development by creation of revolutionized therapies as an alternative
to the classical ones. Even though, at the beginning the immunomodulatory strategies were focused on using CAR-T cells
as an alternative immunotherapy, the latest researches are focusing on developing an alternative immunotherapy such as
CAR-NK. Natural killer cells are an important component of the immune system, having the capacity to secrete cytokinesand chemokines. Because of this property, NK cells can kill the tumor cells and not only. In our review we are going topresent the benefits and the latest results regarding therapies with NK cells, CAR-NK cells, also we are highlighting theimportance of continuing studying them in order to improve the survival of the oncological patients.
Keywords: immunotherapy, chimeric antigen receptor (CAR), CAR-NK, tumor cells.

INTRODUCTION
Even though the conventional strategies used astherapies against cancers such as surgery, radiotherapy,
chemotherapeutic agents offer substantial benefits, thenegative effects of these therapies continue to be an
important challenge [1,2]. Due to this, researchers havefocused on developing new therapeutic alternatives in order
to induce a potent response against tumor cells, with theassociation of fewer side effects [3]. Based on the importantrole of immune system to recognize and eliminate tumor
cells, different immunotherapies have been developed [4].
In the last years, cancer immunotherapy has become an
attractive and revolutionary strategy and showed its
efficiency for treating cancer. This type of therapy utilizes theimmune system in order to induce anti-tumor response byattacking tumor cells through natural mechanisms. For this,researchers have been developed different types of
immunotherapies, many of them being assessed in clinical
trials or being approved by the US Food and Drug
Administration [2,5]. In the table below (Table I), wesummarized all the immunotherapies approaches that havebeen studied till nowadays [6].

Table I: Classification of the immunotherapies
Immunotherapy
Checkpoint inhibitors(CTLA-4, PD1, PDL1)
Agonists ofco-stimulatory signals

Adoptive cell therapy(TILs, TCR, CAR cells)
Vaccines

Cytokines (IL, IFN,
GM-CSF)

Description
class of drugs that regulates
immune system to increase its
response against tumor cells
have the role to bind to specificreceptors present on the T cells
surface and determine in a directway
activation,
growth,differentiation of T cells in order tohave an anti-tumor responsea cell based immunotherapy which
has the role to manipulate immune
cells, with enhancing theirantineoplastic effectcancer vaccines are an alternative
immune therapy that uses dendritic
cells,
tumor
cell
lysate,neoantigens, nucleic acids to inhibit
cancer progression and prevent its
recurrence
these immune regulators have therole to stimulate directly the immune
system for an anti-tumor response

Legend: CTLA4 cytotoxic Tlymphocyte associated protein 4, PD1
programmed cell death protein 1, PDL1 programmed Death ligand
Received April 16th 2019. Accepted May 20th 2019. Address for correspondence: Szekely Flavia, MD, Clinical Emergency County Hospital“Pius Brînzeu” Timișoara, Centre for Cellular and Gene Therapies in the Treatment of Cancer – OncoGen; Liviu Rebreanu Street No. 156, RO300723, Timisoara, Romania; phone: +40726723520; e-mail: flavia_anelise@yahoo.com

Fiziologia - Physiology • 2019.29.1(97)13

1, TILs tumor infiltrating lymphocytes, TCR T cell receptor, CAR
chimeric antigen receptor, IL interleukin, IFN interferon, GM-CSF
granulocyte-macrophage colon-stimulating factor. Adapted from
Riley RS et al. Nat Rev Drug Discov. 2019;18(3):175–196 [6].

From all the immunotherapies we presented above,
Adoptive Cell Therapy (ACT) (Figure 1) have started to gaininterest, in recent years, because of the successful
researches and studies regarding the applications of TIL
therapies for melanoma and solid tumor, TCR-engineered
cells for both hematological and solid tumor, CAR-T cells;
latest being approved by the Food and Drug Administration(FDA) as treatment for B-cell malignancies [7].

 T cell receptor (TCR)-transduced T cell: TCR (Figure 2)
is an alpha beta heterodimer receptor which has theproperty to bind to targeted antigens which areassociated with the major histocompatibility complex
proteins (MHC) [9]. This adoptive cell therapy thatutilizes TCR refers to usage of autologous T
lymphocytes which are modified ex vivo in order topresent T cell receptors specific for the antigens wewant to target.
Chimeric antigen receptor (CAR) therapy: is anotherimmunotherapy which is based on producing, ex vivo,
genetically engineered T cells that express chimeric antigen
receptor. These modified T cells will be able to recognize
tumor-associated antigens [7].

Fig.1. Classification of the adoptive cell therapies
Legend: Schematic representation of the main adoptive celltherapies: chimeric antigen receptor, engineered T cell receptor
and tumor infiltrating lymphocytes. Adapted from Wolf B, et al.
Drug Saf. 2019; 42(2):315-334 [7].

The principle of adoptive cell therapy is to treat the
oncological patients using their own immune cells in orderto have an anti-neoplastic response. For this, different
approaches have been developed, such as: Tumor infiltrating lymphocytes therapy (TILs therapy):
refers to using the lymphocytes infiltration (formed by aheterogeneous population of αβ T cells) presented in
the cellular infiltration from the tumor microenvironment.
This type of therapy refers to infusing back to the patient
his own T cells which infiltrates normally the tumor, after
those cells were isolated and expanded ex vivo. Prior
infusion, the patient undergoes a lympho-depleting
treatment. Even though, at the beginning, TILs weredeveloped as an alternative immunotherapy with
important result in treating malignant melanoma, thelatest researches also showed the utility of using theselymphocytes as a prognostic marker [7,8].

14

Fig.2. T cell receptor
Legend. T-cell receptor has in its structure the α and β chains,
each of them having two extracellular domains, a variable domain
and a constant region. The constant region is associatedtransmembrane with a CD3 complex with three dimers (CD3γ andCD3ε; CD3δ and CD3ε; CD3ζ). For an efficient activation of thecell, also there is required an accessory adhesion molecule
expressed by T cell (CD4 for MHC class II and CD8 for MHC classI). Adapted from Sharpe M, et al. Model Mech. 2015;8(4):337-50[10].

Within this review, we will summarize all therapies
involving the use of chimeric antigen receptor (CAR) andnatural killer cells (NK cells). Finally, we will discuss theutility of a new therapy - CAR-NK- as an alternative
immunotherapy not only for cancer but also for otherpathologies and the latest progresses in using this novel
therapy.

CHIMERIC ANTIGEN RECEPTOR
As we presented above, there are three main adoptive
cell therapies which are currently in research and we are

Fiziologia - Physiology • 2019.29.1(97)going to focus on the CAR cells therapies and their potential
as an alternative immunotherapy.
Structure and generations of CARs
Because of TCR technology limitations to only recognize
intracellular antigens associated with major histocompatibility
complex, researchers developed a non-MHC restricted
method. This new alternative is based on using geneticallyengineered lymphocytes T in order to express on theirsurfaces chimeric antigen receptors which can recognize avary of antigens expressed at higher densities on the cellsurface in a non-MHC-restricted manner. Also, CARs havethe property to recognize other structure such ascarbohydrates, glycolipids, increasing their ability to recognize
a wide range of different targeted cells [11,12].
In 1989 this genetically engineered lymphocytes T
were studied for the first time by Eshhar and his colleagues
at the Weizmann Institute of Science in Israel [12]. They
designed the first generation of CAR, consisted of an
extracellular domain (ectodomain) represented by avariable fragment (scFv) which contain a region for antigen
binding, a connecting sequence – hinge region- between
ectodomain
andtransmembrane
domain,atransmembrane domain and an intracellular domain(endodomain) which has attached a signaling domain thatis derived from CD3 complex (CD3 ζ) (Figure 3). The ScFv
ectodomain of CAR T cell represents the antigenrecognition region and consists of a variable light chain
and heavy chains which are linked by a flexible linker,
designed in recognizing and binding tumor-associatedantigens expressed on the surface of tumor cells [13]. The
most used linker is G4S which confers a series ofproperties such as flexibility and solubility. Also, theorientations of heavy chain, light chain and linker may
influence the affinity and specificity of ScFv [12].

Fig.3. Structure of chimeric antigen receptor (CAR)
Legend. A schematic representation of the chimeric antigen
receptors structure which is made of a variable chain (extracellulardomain), a spacer, a transmembrane domain and an intracellular

Fiziologia - Physiology • 2019.29.1(97)domain (endodomain) which has a variable form depending on the
CAR generation. Adapted from Zhang C, et al. Biomark Res.
2017;5:22 [17].

Between the ectodomain and transmembrane region
there is an extracellular structure called hinge region or
spacer, which has the role to offer stability and flexibility forexpression and activity of CARs. Also, there are researchstudies made by Guest et al., Hudecek et al. which proves
the influence of the spacer over the length and flexibility of
the CARs [14, 15].
The transmembrane region has the role to connect theextracellular and intracellular domains and to offer CAR
stability. It may be formed by a variety of molecules such as
CD3-ζ, CD4, CD8, or CD28. Even though transmembrane
domain wasn't considered an important structure, latest
discoveries showed an influence over CAR-T cell function[13]. Solvado et al. have shown instability over time of first
generation of CAR CD19 whose transmembrane domain
contains CD3-ζ, in comparison with CAR CD19 with CD28transmembrane domain [14]. Morin et al. also demonstrated
the power of extracellular and intracellular CD28 domain topartially induce T cell activation [15, 16].
The endodomain (or the intracellular region) is thefunctional part of the CAR that has the role to transmit thesignal intracellular in order to activate and induce
proliferation over T cell [17]. This intracellular domain is, ingeneral, represented by a CD3ζ domain, but its structure
may differ according to CARs generations [12].
Thus, based on the endodomain structure, there havebeen developed four generations of CARs (Figure 4). The
endodomain of the first generation of CAR is consisted of asingle signaling structure which may derive from IgE(FcεRIγ) or CD3ζ chain. There have been made several invivo studies which demonstrated a better rate in killing tumor
cells for CAR containing CD3ζ endodomain, reason why
almost all CARs that have been developed had in theirstructure a signaling structure derived from CD3ζ [16,17].
Because of the poor outcomes achieved after in vivo studiesof the first generation of CARs properties, researchersdeveloped a second generation of CARs.
The difference between the first and the second
generation of CAR is the presence of a co-stimulatory signal(CD28-B7 system) associated to the CD3ζ domain for thesecond generation of CAR. The CD28/B7 co-stimulatory
domain was added in order to determine a sufficientsynthesis of Il-2 for a better activation of the T cells.
The desire to improve the T cell function and to optimize
the design of CARs, a third generation of this type ofreceptors has been developed. This new generation of
CARs has in their structure a multiple co-stimulatory domain(CD28, CD3ζ, OX40 or 4-1BB) [16].
The latest researches had led to development of a new
and improved generation of CARs (TRUCKs) by using CART cells as a transgenic product carrier for inducing a betteranti-tumor response [18].

15Fig.4. Generations of CARs
Legend. A representation of CARs evolution in all generations thathave been developed. All generations contain an ectodomain, atransmembrane domain and an nedodomain which differs fromgeneration to another.Aadapted from Zhang C, et al. Biomark Res.
2017;5:22 [17].

Production of CAR-T cells
Before the administration of the CAR-T cells to thepatient, there are required several steps in order to obtain
the desired product. From the patient’s peripheral blood,
leukocytes are isolated through leukapheresis. The next
steps involve the separation of T cells from the leukocytes
and their culture activation. Also, it may be performed a T
cells subset CD4/CD8 separation using markers or specificantibody bead conjugates. T-cells isolated from the patient’s
blood are genetically modified using vector (viral or nonviral) (Table II) in order to integrate genetic material which,encode CAR, into T cell genome. This process will lead to
the CARs expression on the T cells surface, cells that will belater expanded in culture in order to have enough cells forinfusing back to the patient [20,21].
Table II. Transfection methods
Viral transduction

Transposons
CRISPR/Cas9
Non-viral transfer
methods- Retroviruses- Adenoviruses- Adeno-associated viruses- Sleeping beauty system- Piggyback system-

Non-viral transfer of plasmid DNA
IVT mRNA

Legend. Classification of all transfection methods used for
inoculation of CARs genome inside the T cells. Adapted from
Miliotou AN, et al. Curr Pharm Biotechnol. 2018; 19(1):5-18 [19].

Clinical applications of CARs
CAR-T cells therapy in blood malignancies
Many clinical trials showed that CAR-T cells therapy
had the most the most effectiveness in relapsed or refractory
B-cell malignancies (B-cell non-Hodgkin lymphoma, acutelymphoblastic and chronic lymphocytic leukemia), with acomplete remission between 70% and 94%. After these16

results, many more trials have been conducted, showing
promising results regarding usage of CAR-T cells therapy
against targets as CD19, CD20, CD30 [19, 22]. The moststudied target is CD19 because of its presence on the B
cells, both normal and neoplasic ones, without being
expressed on other tissues. Even though all clinical trials
showed successful outcomes, CD19 CAR-T cells therapy
induce B cell aplasia, aplasia which may be treated withimmunoglobulin infusion [20,23]. Because of thesesuccessful results, FDA and EMA approved for the regularusage this therapy (Kymriah, Novartis) by using CAR
technology for pediatric patients and young adults
diagnosed with relapse or refractory Acute lymphocytic
leukemia [19].
CAR-T cells therapy in solid tumors
Regarding CAR-T cells therapy for solid tumors, there
are some limitations which make more difficult to treat thesetypes of tumors. Some limitations are represented by: instability of tumor cells by stopping their expressionof the antigens
 histopathological features of the cancer cells
 tumor heterogenicity
 tumor environment which may inactivate CAR-T cells
 inadequate ways of CAR-T cells transportation to thecancer sites.
Many clinical trials showed promising results in using
this therapy for solid tumors by targeting types of proteins(epidermal growth factor receptor, human epidermal growth
factor receptor 2, carcinoembryonic antigen, mesothelin)presented on the cancer cells surfaces, proteins which also
haven an important role in tumorigenesis. In last years,
in vivo/in vitro experiments with T cells transfected withthis type of CARs were able to induce tumor apoptosis[8, 19, 24].

Challenges of CAR-T cells therapy
Even though CAR-T cells therapy has shown impressive
anti-tumor results in clinical trials and in research field, there
are some challenges to overcome in order to obtain a safe
immunotherapy alternative:
 precision in targeting tumor cells
 prevention of mutagenesis and malignant transformation
during viral vector transduction (genotoxicity)
 a better control of the activation and proliferation of CART cells
 prevention of side effects caused by CAR-T cell infusion:‐ neurotoxicity‐ cytokine release syndrome (CRS)‐ B cell aplasia (CD19 CAR-T cell therapy)
Fiziologia - Physiology • 2019.29.1(97)‐Systemic inflammatory response syndrome[19,23].

NATURAL KILLER CELLS
In order to have a better knowledge of CAR-NK, first, wemust understand the biology and role of the natural killer
cells, as well as their clinical applications; subjects we aregoing to approach in this chapter of our review.

NK cells have their unique ability to recognize target
cells that don’t express major histocompatibility complex(MHC) molecules on their surface [27]. Besides this ability,
NK cells have a potent cytotoxic activity by releasingproteins (perforin, granzymes), can secrete cytokines andchemokines in order to regulate the immune system
response [30]. Because of all these properties and the abilityof fast killing, makes NK cells an important candidate for
adoptive cell therapy [30].

Preparation of NK cells for immunotransfer
The biology and role of the natural killer cells
Natural killer cells (NK cells/killer cells) are large
granular lymphocytes which, together with eosinophils,
basophils, phagocytic cells, and mast cells are part of the
innate immune system and they also have an important rolein defense against cancer and viral infections with alimitation of their spread. These cells originate from bonemarrow, developing from the hematopoietic progenitor cells(HPCs) – CD34+. Latest researches show that NK cells may
also develop in secondary lymphoid tissues such as lymph
nodes, tonsils, spleen and this is highlighted by thepresence of NK cells subset CD56 bright – high density
surface expression of CD56, which can be isolated fromsecondary lymphoid tissue [25-27].
According to their function and immunological phenotype,natural killer cells are divided in two main subsets:
CD56dimCD16bright and CD56brightCD16dim, CD56 andCD16 being the NK cells surface antigens [25,28]. 90% of
circulating NK cells express on their surface CD56dim and in
response to target cell stimulation, they have the property tomediate cytotoxicity. The rest of the NK cells express
CD56bright and have a greater ability to both secret andrespond to cytokines. In comparison to lymphocytes (B and
T), natural killer cells don’t express on their surface specificreceptors for different antigens. Thus, when an NK cells
encounter a tumor cell which does not express a self MHC
class I, there are induced several mechanisms in order to kill
the tumor cell such as: expression of FasL or TNF- related
apoptosis- inducing ligand (TRAIL) which induce death oftumor cells by interacting with their receptors (Fas and TRAIL
receptors), releasing of cytoplasmic granules which contains
granzymes and perforins.
Besides the presence of CD56 and CD16 on their surfaces,
functionally, natural killer cells also express receptors thatclassified them as activating and inhibitory. Also, the interaction
with other immune components may activate or inhibit NK cells
activation and function. Cytokines (IL-2, IL-15, IL-12, IL-18),
interactions with macrophage, dendritic cells, stromal cells
have role in regulation of NK cell function. In contrast,
secretion of transforming growth factor-beta (TGF-β), or
IL-10 produced by acute myeloid leukemia (AML) are known
to inhibit activation and function of NK cells [28].

Fiziologia - Physiology • 2019.29.1(97)Methods regarding isolation, expansion and enhancing
are necessary in order to use natural killer cells as atherapeutic source. Isolation and production can be done on
NK cells obtained from different sites (Table III) [32].
Table III: Classification of potential NK sources
Umbilical cord blood
Adult lymphapheresis products
NK cells lines
Induced pluripotent stem cells (iPSC-NK)
Human embryonic stem cells (hESC-NK)
Legend. Classification of available resources used for isolation ofnatural killer cells. Adapted from Darji A, et al. J Stem Cell Res
Ther. 2018; 8(3) [25].

First, leukocytes must be isolated through leukapheresis.
After this process, with the help of CD56 beads, NK cells are
isolated and then, infused back to the patient. In order tostimulate their expansion and activation, systemic cytokines(usually IL-2) are administrated.
Another alternative to autologous NK cells is the use ofallogenic ones, over the last years being considered aspromising alternative immunotherapy. Miller and his team
were among the first researchers who demonstrated that
this type of ex-vivo expansion of haploidentical NK cells andtheir administration after a lympho-depleting chemotherapy
is a safe alternative without the risk of inducing graft vs hostdisease (GVHD) [28].
The latest approaches have focused on alternative ways
to maintain and stimulate activation and expansion of NK
cells. After the NK isolation, these cells must be expended
and activated in culture cells medium enriched withcytokines such as IL-2, IL-15, IL-18, IL-21, type 1 Interferon.
When will be an adequate number of cells, the patient will
be infused with those cells [25,28,31].
More recent, researchers focused on developing
alternative methods in order to produce larger quantities ofpurified and functionally active NK cells by using “feeder
cells” such as: monocytes in the form of irradiated PBMCs,
EBV-transformed lymphoblastoid cell lines, irradiated K562
cell that express membrane-bound IL-15/IL-22 and 41BB

17

ligand for a co-stimulation in gas-permeable expansion
flasks [28-32].

Clinical applications of NK cells
When it comes to utilization of NK cells as a source of
immunotherapy, several preclinical and clinical studies
demonstrated the viability of NK cell lines to be used asa prognostic marker and therapeutic treatment in cancer[25-26].
There are evidences (experimental and clinical) whichsuggests the possibility of using NK cell as a prognostic
marker in cancer. An 11 years study shows that low levelsof NK in peripheral blood lymphocytes are correlated with anincreased risk for cancer and the absence/low levels ofthese cells in the tumor infiltrating lymphocytes is a positive
prognostic marker in multiple malignancies and theintratumoral presence of NK cells are correlated withimprove outcome and delay of tumor progression [26, 28].
When it comes to hematological malignancies, there are
multiple studies which suggest using NK cells as analternative therapy, being associated with successful
outcomes. All these results are proving its effectivenessunlike T-cell therapy [26]. In vitro were obtained the mostsuccessful results of using NK cells in different malignancies
such as acute myeloid leukemia (AML), acute lymphocytic
leukemia (ALL), multiple myeloma (MM) [30]. Also, same
results were obtained for solid tumors (neuroblastoma,
ovarian, renal cell, colon, gastric carcinoma) [25].
Even though natural killer cells may be a promisingtherapeutic alternative, for the treatment of solid tumors
there are some challenges to overcome:- Development of transport support of NK cells to thetumor site
- Ways of tumor capsule penetration in order to exert
their function- Tumor targets must be susceptible to NK-mediatedcytotoxicity- Altering the tumor microenvironment which inhibits NK
cells function- Finding antigen specific for each tumor cells [28].

CAR-NK
Using NK cells in order to express chimeric antigen
receptor is an innovative immunotherapy, developed as anecessity for a better improvement of the NK cell clinicalefficacy [32].
In the last past years, researchers have made importantdiscoveries regarding genetic engineering immune cells
used as therapy for treating cancer and not only. First, havebeen extensively used and studied the clinical potential of
CAR-T cells, being successfully used in treating leukemia
and lymphoma. But this novel therapy it is associated withseveral side effects and limitations, researchers focused infinding alternative immune cells in order to express chimericantigen receptors in order to overcome this challenge.
Because natural killer cells can kill their targets in a nonspecific manner and are cytotoxic immune effectors, thelatest researches have been done on using this type of cells
as a base to express chimeric antigen receptors [28].
As we know from the previous chapter, natural killer cells
are effectors of the immune system with a cytotoxic effectagainst tumor cells. Besides the cytotoxic effect, NK cells
have multiple functions such as: degranulation, cytokine
release, functions which are controlled by different signals
from inhibitory receptors, activating receptors andheterodimeric C-type lectin receptor (NKG2A) with role inrecognizing ligands present on the surface of targeted cells[30].
Because of their functions and efficiency NK cells havebeen used as an alternative for the classic oncologicaltherapies [28, 32].
Only in preclinical tests have been demonstrated thefeasibility of natural killer cells genetically engineered in order
to express CARs, primary NK cells and NK-92 successfully
expressing chimeric antigen receptors against several targets
such as: CD19, CD20, CD44, Her2. In vitro and in vivo, CARNK cells efficiently mediate the killing of tumor targets. Also,
Shimasaki and his team succeeded in testing expression of
CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in
human NK cells using mRNA electroporation as an alternativetransfection, with the appearance of transfection result in 24
hours. Also, the levels of CARs expressed on NK cells weresimilar with levels of CAR-NK achieved through retroviraltransduction [30].

Other pathologies
Though, NK cells therapy has been developed as atherapeutic alternative in different types of tumors, there areclinical trials which demonstrate the usage of this therapy
with NK cells in viral infections (hepatitis, influenza, HIV).
Also has been studied the role of natural killer cells in otherdiseases like asthma, diabetes, arthritis, multiple sclerosis.
All these researches prove these cells, may be used as apotential therapy for other pathologies but it is necessary abetter understanding of function and receptors of naturalkiller cells. [25].

18

CAR-NK therapy
The major advantage of using CARs is the possibility ofequipping immune cells with the ability to recognize and killtheir targets that are associated with MHC [32].
There are a series of advantages that shows thesuperiority of CAR-NK compared to CAR-T cells (see Table I)- Autologous and allogenic natural killer cells have alimited persistence in vivo

Fiziologia - Physiology • 2019.29.1(97)Low risk of life-threatening toxicity: cytokines release
syndrome (CRS) because of the short rate of
expansion of clonal NK cells and the immune
mediated rejection of allogenic natural killer cells- Preclinical and clinical trials have showed that NK
cells do not cause GVHD
- The possibility to produce an off-the-shelf allogeneic
product which may be used for immediate clinical use
When it comes to safety of the method, same concerns
as for CAR-T cells are still available. Cytokine release
syndrome, toxicity to normal tissues, on-target/off-tumors
effects, tumor lysis syndrome, GVHD are relevant regarding
the safety of CAR-NK therapy for oncological patients.
Another challenge is genetic manipulation of NK cells. Evan
though viral transduction is successful for T cells, unfavorable
effects have been noticed on cell viability of transduced NK
cells, reason that lead to optimization of viral transduction andelectroporation technologies. Although all these problems
regarding CAR-NK cells needs to be improved in order tohave a safe and efficient therapy alternative, the biggest
challenge remain finding the optimal target appropriate
antigens specific for each tumor cells [30,32].
The main sources such as peripheral blood (PB), cord
blood (CB), hematopoietic stem cell progenitors (HSCPs)
are used for isolation of natural killer cells, cells which havebeen successfully genetically engineered to express
chimeric antigen receptors [28].
-CAR-NK cells in Leukemia and Lymphoma
In preclinical researches and clinical trials, have beenstudied the anti-tumor activity of CAR-NK cells that wereengineered to target specific antigens such as CD19, CD20,
CD33, CD138, CD319, CD3, CD5, CD123. From all theseantigens, CD19 is the most studied and, in 2004 waspresented data regarding NK-92 cell line harboring antiCD19-CAR. Later, in 2016 were published results showing
an increased cytotoxicity of anti-CD19 CAR-NK92
compared to unmodified NK92.
In acute myeloid leukemia studies highlighted thepossibilities of targeting CD33 which is expressed by both
healthy and malignant myeloid cells. Several approaches
such as CD33-directed CAR-modified T cells with a scFV
derived from gemtuzumab ozogamicin, CD33-directed NK92 have been studied, both of them showing a potent
cytotoxic activity against targeted cells. Because of CD123
expression in a high rate in malignant hematological cells
and at low levels for hematopoietic stem cells, researchers
focused on developing a therapy by targeting this antigen.
Despite of its potential, anti CD123 CAR-T cells induced
severe hematological off-target toxicities in mouse model.
Therefore, have been developed an alternative to anti
CD123 CAR-T cells - NK cells expressing anti CD123 CARs

Fiziologia - Physiology • 2019.29.1(97)- which had shown in vitro activity against acute myeloid
leukemia cell line and patient sample [33].

Solid tumors
Regarding multiple myeloma, there are studies showing
evidence of efficiency of NK-92 cell line as a potential therapy
in treating this type of cancer. Also have been successfully
tested in vivo/in vitro anti CD319 and anti-CD138 CARs
which were inserted into NK-92 and NK92MI (it is an NK92
IL2-independent) against primary and cell lines of multiple
myeloma cancer cells. Recently, researchers are taking inconsideration using SLAMF7-directed CAR-NK cells
combined with CD38-directed antibody daratumumab as analternative therapy for relapsed multiple myeloma.
In the past and present, it has often been shown that theNK-92 cell line can be effectively transduced with several
different CARs against several malignancies for testing in
preclinical approaches and currently in first clinical studies.
CAR-NK-92 cells were quite successful in overcoming thetumor barrier and retargeted anti-tumor cytotoxicity against
several resistant solid tumors, including epithelial cancers bytargeting of human epidermal growth factor receptors (HER1[ErbB1], HER2 [ErbB2]), neuroectodermal tumors by GD2,
brain tumors by HER1 and HER2, and ovarian carcinomas
also by HER2 [4, 6, 28]. However, there are some limitations
to using this cell line [33].

THE FUTURE OF CAR-NK CELL THERAPY
The latest researches regarding a better understandingof NK cell biology and immunological function have paved
the way to development of novel immunotherapies
alternatives [1]. In the next years, there will be a greaterinterest regarding the improvement of tumor-specific NK
cells efficiency, ofgene editing techniques with
developing better alternatives (CRISPR/Cas9, TALEN),
targeting the tumor microenvironment, inhibiting the immune
checkpoints and developing delivery biomaterials [2,3].
Although pre-clinical tests of CAR-NK therapy as
potential therapy have begun in recent years, the promising
results encourage continued testing and a later applicationof this therapy in clinical trials [4].
In this review we discussed the importance of adoptive
cell therapies, the biology and function of chimeric antigen
receptors, of natural killer cells and CAR-NK cells, as wellas the importance of increasing focus on clinical applications
and improvement the practicality of these geneticallyengineered immune components.
Acknowledgement: This work was supported by the grant“Chimeric Antigen Receptor Targeted Oncoimmunotherapy with
Natural Killer Cells (CAR-NK)”, POC 92/09/09/2016, ID: P_37_786,
MySMIS code: 103662.

19

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Fiziologia - Physiology • 2019.29.1(97)TERAPIA CU CELULE CAR-NK: ERA UNEI NOIPOTENȚIALE IMUNOTERAPIIREZUMAT
Domeniul imuno-oncologic se află într-un proces continuu de dezvoltare prin creearea de terapii revoluționare ca oalternativă la cele clasice. Chiar dacă, la început, strategiile imunomodulatoare s-au axat pe folosirea celulelor CAR-T cao alternativa imunoterapeutica, cele mai recente cercetări se concentrează pe dezvoltarea unor terapii alternative cum arfi CAR-NK. Celulele natural killer sunt o componentă importantă a sistemului imunitar, având capacitatea de a secreta
citokine și chemokine. Datorită acestor proprietăți, celulele NK pot ucide celulele tumorale si nu numai. În cadrul reviewului nostru, vom prezenta beneficiile si cele mai recente rezultate legate de terapiile celulare cu NK, CAR-NK cele mai
recente studii in ceea ce priveste terapia cu celulele NK, celulele CAR-NK, de asemenea punem in evidenta importanța
continuării studiului lor pentru a îmbunătăți rata de supravietuiere a pacientilor oncologici.
Cuvinte cheie: imunoterapie, receptorul antigenic chimeric (CAR), CAR-NK, celule tumorale.

Fiziologia - Physiology • 2019.29.1(97)21NEURODEGENRATION AND NEUROPROTECTION
IN SEVERE RETINAL DYSFUNCTIONISTRATE BOGDAN MARIUS1, VILCIU CRISANDA2
1University Hospital Antwerp, Belgium, Neuroscience Center
2Clinical Institute Fundeni, Bucharest, Romania, Department of Neurology

ABSTRACTThe retina is a very complex structure, consisting in a thin multilayer of primarily neural cells derived from ectoderm. The
retina can be assimilated as a photo-camera being highly specialized in perception, capture, signal initiation andtransmission together with the visual stimuli from the “outside world “to the optic nerve and finally to the visual cortex of thebrain. As a highly specialized ultrastructure with its very complex function, the pathological events in the retina can be
schematized as: retina-visual pathway-photoreceptors-phototransduction-ganglion cells, horizontal cells (Amacrine cells)interplexiform neurons. All those microstructures get affected by different aggressor either internal or external leading in
90% of the clinical situation to the vision loss. The present article tries to summarize the cellular and molecular mechanism
in retina degradation and disfunction, as well by suggesting modern approaches and the contribution of someneuroprotective therapies in maintaining the optimal physiologically role of the retina in some retinal pathological conditions.
Retinal neurodegeneration is an irreversible process which should be seriously taken into account having in view that,
unfortunately, the corneal, macular or uveal degenerescence of disfunction can be partially solved, the degenerescence ofthe retina stays extremely delicate to be treated despite of all modern technology and therapeutically options. Retinaldegeneration is a progressive neurological disorder caused by genetic mutations and/or environmental or pathologic
damage to the retina; unfortunately, the problem is incurable.
Keywords: visual function, retina, neurodegeneration, retinal vascular degeneration, neuroprotection.

INTRODUCTION
Neurodegeneration plays a major role in the retinal
dysfunction, which is still thought to be the main
determinant of permanent disability, either minor,
moderate or severe. The visual function stays a primordial
one even during the different clinical stages of vitreous,uveal, macular and retinal damage. To foresee, to preventand to treat any type of injury at these ocular levels, thecellular and molecular alteration are a very important
prerequisite. Cellular dysfunctions occurred due to thechanges in the retinal microenvironment lead from clinicalcomplication to clinical complication and finally toblindness. The vascular compound of the retina should
also be outlined as a contributor to all retinal pathologies.
The administration of anti-vascular endothelial growth
factor (anti VEGF) to suppress the vascular proliferationand exudation has highly impaired the prognosis of some
unpleasant events [1].

Cellular disorders in the retina in a nutshell
It is very important to have a critical view on allcompromising factors that plays a role in retinal
neurodegeneration implying the severe dysfunction at theretinal level. We try briefly to illustrate the main attributes
which should arouse attention: exogenous and endogenous.
The synergic actin between those two factors and in
between neural cells (retinal neurons and Mūller glial cells)
via intracellular signaling, can cause, of course, neuralfunctional disorders, abnormal gene expression andapoptosis. In this regards the retinoblastoma and as wellother genetic condition of the eye like Retinal dystrophy with
inner retinal dysfunction and ganglion cell anomalies stays
the most life-threatening factor. Intracellular-inflammatory
signals and ROS (reactive oxygen species) can be activatedsimultaneously: endogenous/exogenous. The pathologicaland disrupted generated signal will proliferate in all retinal
structure conferring a maladaptive function by debilitating intime the patient. In the cases of elderly retinitis pigmentosa

Received March 16th 2019. Accepted May 20th 2019. Address for correspondence: Istrate Bogdan Marius, MD, PhD, University Hospital Antwerp,
Belgium, Neuroscience Center, Wilrijkstraat 10, 2650 Edegem, Belgium; phone: +32 3 821 30 00; e-mail: istratem.bogdan@yahoo.com

22

Fiziologia - Physiology • 2019.29.1(97)and retinoblastoma, ROS can generate de activation ofmediatory intracellular effectors, such as: MAPK (mitogenactivated protein kinase), STAT (signal transducer activationof transcription proteins) and NF-kB (activated protein
complex). If the condition is genetic, the intimate mechanism
are very complex leading as well to a problematic clinicalstrategy sometimes.
Interleukin 6 (IL-6)-mRNA is expressed in the retinal
ganglion cells. Pro-inflammatory signaling pathway can also
exert a negative effect on retinal micro -vascularization. The
retinal angiogenesis is also perturbed at this level. Retinal
ganglion cells, expressing cytokines such as VEGF [2], areresponsible also in an important condition called stressinduced retinopathy.
Most ocular diseases cause harmful neurodegenerative
processes by expressing Gap 75 leading to thephotoreceptors atrophy, cell death and impacting seriously
the visual function. Calcium-gated voltage channels are also
involved. The signaling cascade of the molecular events of
Ca2+ channels antagonist D-cis diltiazem, disrupts the
kinetic of rd 1 rod alteration, announcing the rescue of the
scotopic vision [3].
Breakdown of the blood-retinal barrier
At the cellular and molecular level, the damage of bloodretinal barrier can be seen only post-mortem by anatomopathological remarks after the Evans blue test necessary tobe performed to appreciate the blood-retinal barrier
permeability. An immune adaptive response will be also
triggered by the activation of the microglia/macrophages
pathway which will destabilize the blood-retinal barrier
favoring in this way moderate to severe hemorrhage. The
demyelination of the optic nerve should be also mentioned
and prevented by administrating of neuroprotective andneurotrophic factors.
Intracellular metabolic consequence in retinal cell deathand the inflammatory burst can also lead to uveitis and agerelated macular degeneration. During the retinal metabolic
stress, the released glutamate will cause the death ofneurons containing ionotropic glutamate with a negative
consequence as an insufficient supply of nutrients to theoptic nerve, uvea, and retina.
Retinal ganglion cells (RGC), need also to protect(neurotrophic/neuroprotective agents) the retinal pigment
epithelium. There is no need to outline that RGCs are avariety of a very vulnerable population. RGCs own a highenergy demand, but in contrast to the retinal
microvasculature, their network and structure is not sodense like the ones in the brain. On the other hand, we cantalk about a natural compromise between the metabolic
demands of the neurons and a optimal vascular supply [4].
In the pediatric population retinopathy of prematurity, andretinoblastoma claims an irreversible damage of all ocular
microvasculature and retinal cells destruction.

Fiziologia - Physiology • 2019.29.1(97)Fig.1. (A) Fundus photograph. An arrow shows the center of theretina, where photoreceptor cells and light stimuli are concentrated.
(B) Optical coherence tomography (OCT) image showing a crosssection of the central part of the retina. NFL, nerve fiber layer; OPL,
outer plexiform layer; ONL, outer nuclear layer (photoreceptor cell
layer); OS, outer segment. (Adapted after Ozawa, Sasaki et al.
Current Pharmaceutical Design, 2012; 18: 51-56).

Other physiological attributes of the retinal cytoarchitecture impacting visual function and pathology
In the retina structure we found also multiple layers ofinterconnected neurons and also in the context of visualfunction, fovea plays an important role as well. Fovea is acentral region of the human and primate retina consisting ina high number of cones cells responsible for an excellent
visual acuity and the chromatic sense. Age-related maculardegeneration (AMD) is a reversible loss of vision, the age
being the most incriminated risk factor. Other metabolic
conditions, such as: diabetes mellitus, hyperlipidemia,
hypercholesterolemia, hyperuricemia, higher systemic
blood pressure also affect the uveal and retinal
microvasculature path. Cellular responses induced byretinal injury: alternations and dysfunctions in the retinal
neurons and circuits, changes in photoreceptors and theirsynaptic connectivity, and here the clinician either aneurologist or ophthalmologist should be aware of thedisease Rhodopsin-positive neuritis associate with gliotic
Müller cell processes. The cell morphology and function as
well the clinical features will significantly differ from a healthy
individual to an individual in ill conditions.

Fig. 2. Cellular remodeling during retinal degeneration. Schematic
representation of the main changes in morphology (A) and23

connectivity (B) that take place in retinal neurons during
degenerative processes, regardless of the origin of the damage. (A)Signs of rod and cone degeneration are the reduction in the size ofphotoreceptors, the shortening of both outer and inner segments,
and the loss of synaptic connections with second-order neurons.
Bipolar cells display early retraction and loss of dendrites during
retinal degeneration, with further sprouting of ON rod bipolar cell
dendrites into the ONL in some degenerative diseases. In
advanced degenerative processes, the retraction of dendrites
may also occur. The axonal endings of bipolar cells areshortened. Horizontal cells retract their dendrites during retinaldegeneration, although the sprouting of dendrites and axon
terminals are frequent, with the formation of ectopic synapses in
the ONL. Remodeling of AII amacrine cells involves the loss oflobular appendages in the OFF strata of the IPL in several retinaldegenerative diseases. (B) Summary of the connectivity changes
occurring in retinal neurons during the course of the degenerative
process at the OPL. Rod bipolar cells make new contact with theremained cones. (Adapted after N. Cuenca et al. Progress in
Retinal and Eye Research, 2014; 43)

Defragmentation and destruction (shortening of synaptic
ribbon) are also threatening factors leading to retinal
detachment. During the retinal damage a cellular
remodeling may took place but needs to be still elucidated
and defined.
Oxidative stress contributes as well to the retinaldegeneration because retinal protein homeostasis
modulates the degeneration process but, on the other handregulates the action of some neuroprotective agents with an
impact on retina, vitreous and optic nerve.
Therapeutic neuroprotective therapies
1.
2.
3.
4.
5.
6.
7.
8.

24

Anti-apoptotic therapy
Anti-growth-factor vascular extravasation therapy
Norgestrel
Antioxidant cocktails
Ginkgo biloba extract
Resveratrol
Quercetin
Cell-based therapies

9. Surgical procedures (laser Ar/Ne, Cryoprecipitation, Cry ocoagulation)
There are some others natural adjuvant therapeutic
options but the clinician can make an accurate selection todecide which patient can benefit of it and the optimal
dosing/time effect.

CONCLUSIONSHuman retinal degenerative diseases are currently
incurable and retinal degeneration, once initiated, isirreversible. Cell replacement through the transplantation ofstem cell or differentiated retinal cell types and artificial
vision were as well taken into account and there are clinical
trials win those regards. Anyway, despite the efforts ofresearchers to identify a therapy capable of preventing
retinal degeneration or restoring vision, current therapies
entail difficulties that need to be addressed to achieve safe,
effective treatments. In this context, the administration ofantioxidants (alone or in cocktails), anti-apoptotic, antiinflammatories, neurotrophic factors or viability factors
unfortunately only slows the neurodegeneration of the retina
by delaying retinal cell death, but fail to prevent the
progression and prognostic of the disease.

REFERENCES
1. N. Cuenca et al. Progress in Retinal and Eye Research, 2014;43.
2. Ozawa Y, Kamoshita M, Narimatsu T, Ban N, Toda E, et al.
Neuroinflammation and Neurodegenerative Disorders of theRetina. Endocrinol Metab Synd., 2013; 2: 111.
3. Esmaeelpour M, Považay B, Hermann B, Hofer B, Kajic V, etal. Mapping choroidal and retinal thickness variation in type 2
diabetes using three-dimensional 1060-nm optical coherence
tomography. Invest Ophthalmol Vis Sci., 2011; 52: 5311-5316.
4. Ding X, Patel M, Chan CC. Molecular pathology of age-related
macular degeneration. Prog Retin Eye Res., 2009; 28: 1-18.

Fiziologia - Physiology • 2019.29.1(97)NEURODEGENERAREA ȘI NEUROPROTECȚIA
ÎN DISFUNCȚIILE SEVERE ALE RETINEIREZUMAT
Retina este o structură foarte complexă, constând într-un multistrat subțire de celule neuronale primare derivate din
ectoderm. Retina poate fi asimilată ca o cameră foto fiind extrem de specializată în percepție, captare, inițierea semnaluluiși transmitere împreună cu stimulii vizuali de la „lumea exterioară” la nervul optic și în final la cortexul vizual al creierului.
Ca o ultrastructură extrem de specializată, cu funcția sa foarte complexă, evenimentele patologice din retină pot fi
schematizate ca: cale retină-vizuală-fotoreceptori-fototransducție-ganglion celule, celule orizontale (celule amacrine) neuroni interplexiformi. Toate aceste microstructuri sunt afectate de un agresor diferit, fie intern, fie extern, ceea ce duce la
90% din situația clinică la pierderea vederii. Prezentul articol încearcă să rezume mecanismul celular și molecular în
degradarea și disfuncția retinei, precum și prin a sugera abordări moderne și contribuția unor terapii neuroprotectoare lamenținerea rolului fiziologic optim al retinei în unele condiții patologice ale retinei. Neurodegenerarea retinei este un proces
ireversibil, care ar trebui luat serios în considerare, având în vedere că, din păcate, degenerescența retinei,corneei, maculei
sau uveii poate fi parțial rezolvată, degenerescența retinei rămâne extrem de delicată pentru a fi tratată în ciuda întregii
tehnologii moderne și opțiuni terapeutice. Degenerarea retinei este o tulburare neurologică progresivă cauzată de mutații
genetice și / sau de deteriorarea mediului sau a patologiei retinei; din păcate, problema este incurabilă.
Cuvinte cheie: funcție vizuală, retină, neurodegenerare

Fiziologia - Physiology • 2019.29.1(97)25TUBERCULOUS PLEURAL EFFUSION OR
DEXTROPOSITION OF THE HEART? A CASE REPORTDRAGOS NICOLOSU1, MARIUS GEORGESCU2, RAMONA CIOBOATA3
1Pulmonology Department - Victor Babes Clinical Hospital, Craiova, Romania
2Physiology Department, University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania
3Internal Medicine - Pulmonology Department, University of Medicine and Pharmacy Craiova, Romania

ABSTRACT
Pleural effusions are commonly encountered in clinical practice. Cardiac dextroposition is the horizontal displacement of
the heart into the right hemithorax. Present case study is of an unknown congenital cardiac dextroposition, a female patient,aged 61 years, with no history of pulmonary or cardiovascular pathology, who was admitted with a suspicion of tuberculous
pleural effusion. Chest X-ray (poster anterior view) showed the presence of a homogenous opacity occupying the half lower
part of the left hemithorax, an image of hyperlucency rounded of a thick wall located next to the left hilum with themediastinum shifted to the opposite side.
Keywords: tuberculous pleural effusion, dextroposition of the heart, chest X-ray.

INTRODUCTION
Pleural effusions are a common sight in clinical practice,for both respiratory specialists and others. [1] Identifying thecause for pleural effusion can be achieved in the majority ofcases through a combination of physical examination,
analysis of relevant clinical history, chest radiography anddiagnostic thoracentesis. [2] Tuberculous pleural effusion
constitutes the 2nd most common form of extra pulmonary
tuberculosis, and in endemic tuberculosis areas it
represents a frequent cause of pleural effusions. It is usuallypresented in acute form, with the most common symptoms
being dyspnoea, fever, cough, and pleuritic chest pain. [3]The definition of cardiac dextro-position is the presenceof the heart, with the major axis of the heart in normalalignment, in the right hemithorax. No anatomic alteration ispresent in the heart itself, but rather only in its location.[4]The dextroposition of the heart can be caused either by
the congenital malformation of the organ, or of the tractionof the organ over to the right side by extraneous mechanicalcauses such as tumors, the effusion into the left pleura,traction of right-sided pleuritic adhesions or cystic diseaseof the left lung.[5]Dextroposition of the heart is different from true
congenital dextrocardia, in which the apex of heart is pointed
towards the right, and is formed by the left ventricle. In

dextro-position the location of the left atrium and ventricle ison the left side of the heart chambers. The present clinicalcase showcases an unusual cause for cardiac
dextroposition.

CASE REPORT
A female patient, aged 61 years, with no history of
pulmonary or cardiovascular pathology, was referred to theclinic by the family doctor with a diagnostic of tuberculous
pleural effusion under observation. She was admitted forphysical asthenia, non-productive cough and dyspnoea.
Patient reported ongoing symptoms of mild nonproductive cough and dyspnoea over the preceding 4 weeks.
She is a current tobacco smoker with a 15-pack-years
history and denied drug usage or travel abroad in thepreceding year. The patient’s son was admitted to the clinic 3
months previously, with a diagnosis of pulmonary tuberculosis.
On examination, the patient was discovered to be ofnormal weight (IMC value of 22) with the following vital
parameters values: a body temperature of 37.8 Celsius
degrees, a heart rate of 89 beats/minute, respiratory rate
16/minute, blood pressure was 140/85 mmHg, and oxygen
saturation measured by pulse oximetry was 98% on room air.

Received March 14th 2019. Accepted May 31st 2019. Address for correspondence: Marius Georgescu, MD, PhD, Assistant Teaching Professor,
Department of Functional Sciences, Discipline of Physiology, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu Square No.
2A, Timisoara, RO-300041. Phone: 0040765527318; e-mail: georgescu.marius@umft.ro26

Fiziologia - Physiology • 2019.29.1(97)Palpation revealed reduced vocal resonance on the halflower part of the left hemithorax, dullness was detected on
percussion over the same area and auscultation revealed
diminished vesicular breath.
Laboratory investigations performed, including full bloodcount, amylase determination, liver function tests, urea andelectrolytes determination, were all within normal limits. TBinterferon γ release assay (TB-IGRA) result was positive.
Thoracic radiography (poster anterior view) revealed
the presence of a homogenous opacity occupying the halflower part of the left hemithorax, an image of roundedhyperlucency of a thick wall located next to the left hilum withthe mediastinum shifted to the opposite side (Figure 1).

Fig. 3. Computer tomography showing bilateral fibrosis lesions,bilateral inferior lobular cylindrical bronchiectasis, without pleuralor pericardial effusion.

Based on the clinical, biological, imagistic chart, thediagnosis of dextro-position of heart was established.

DISCUSSION

Fig. 1. Chest x-ray showing the presence of a homogenous opacity
occupying the lower half part of the left hemithorax, an image ofrounded hyperlucency of a thick wall located next to the left hilum
and the mediastinum shifted to the opposite side.

Based on anamnesis, clinical examination, laboratory
analysis, at the time of the investigation the diagnosis
suspicion of tuberculous pleural effusion was raised.
Thoracentesis was attempted, the result was negative forthe diagnosis suspicion, therefore computer tomography ofthe chest and abdomen was performed.
Computer tomography revealed bilateral fibrosis lesions,bilateral inferior lobular cylindrical bronchiectasis, without
pleural or pericardial effusion, ascension of the left
hemidiaphragm, stomach and intestinal cords visible in thepostero-inferior mediastinum (Figures 2 and 3).

Fig. 2. Computer tomography showing, the ascension of the left
hemidiaphragm, stomach and intestinal cords visible in thepostero-inferior mediastinum.
Fiziologia - Physiology • 2019.29.1(97)Pleural effusion represents an abnormal accumulationof fluid within the pleural space. It does not constitute a
disease per se, but rather the complication of another
underlying pathology [6]. The excess of fluid results from an
overthrow of the equilibrium which is present across thepleural membranes. Thus, pleural effusion constitutes anindicator of a pathologic process, which can be of primary
pulmonary origin, it can originate in another organ system,or it can be tied to a systemic disease. It is not a diagnosis
in and of itself, and it can occur within a setting of either
acute or chronic disease. Of the forms of extra-pulmonary
tuberculosis, tuberculous pleural effusion represents one of
the most common ones.
The patient was referred to the clinic, with a diagnosis ofa possible tuberculous pleural effusion, since she was
identified as a contact in the epidemiological investigation ofher son - admitted to our clinic 3 months previous with adiagnosis of pulmonary tuberculosis.
Following the investigation, the diagnosis was negatedby the absence of fluid on computer tomography and theascension of the left hemidiaphragm, stomach and intestinal
cords visible in the postero-inferior mediastinum and dextroposition of heart.
Dextro-position of the heart is classified as congenital or
acquired. Congenital dextroposition of heart, in association
with other cardiopulmonary anomalies, constitutes scimitar
syndrome [7], with hypoplasia of the right lung [8]. Acquired
dextro-position of heart is often seen in patients who
underwent pneumonectomy, due to a multitude of factors:
hyper expansion of the non-operated lung, the collapse of thepleura depending on the retraction of the intercostal space,the obliteration of the post pneumonectomy space which
depends on both the reabsorption and the organization of thefluid it contains, elevation of the diaphragm.

27

In the present case, the suspicion is that the cause of
the dextro-position of heart is congenital, since the patient
had not underwent a cardiovascular examination or even a
chest radiography ever before. However, none of the otherusual cardiopulmonary anomalies were present. Thus, theexplanation for dextro-position in the present case is most
likely be due to elevation of hemidiaphragm with mediastinal
shift.
Patients suffering from cardiac dextroposition are
generally asymptomatic [9]. In the present case, thepatient was admitted for dyspnoea, physical asthenia andnon-productive cough, without any past medical history
present.
Routine physical examination revealed the cardiac
impulse identifying the cardiac apex closer to the sternum.
Percussion performed on the heart borders confirmed thedisplacement of the heart to the right [10].
Chest radiography (poster anterior view) identified the
cardiac silhouette, which was displaced to the right. This cansometimes be associated with the presence of chest wall
deformities, or various other pathologies present in thediaphragm and the lungs.
In the present case, the chest X-ray revealed thepresence of a homogenous opacity occupying the half lower
part of the left hemithorax, an image of roundedhyperlucency of a thick wall located next to the left hilum withthe mediastinum shifted to the opposite side. This raised thesuspicion of pleural effusion, however, since thoracentesis
was negative, further investigations were necessary.
Computer tomography was performed, revealing ascensionof the left hemidiaphragm, stomach and intestinal cords
visible in the postero-inferior mediastinum and the heart
displaced to the right.

28

CONCLUSION
Cardiac dextroposition represents the simple horizontal
displacement of heart, to the right side of the chest, ofcongenital or acquired causes. The most frequent cause ismechanical, with acquired dextro - position of heart mostcommonly present in post-pneumonectomy patients.
Patients suffering from cardiac dextroposition are generally
asymptomatic. The diagnosis is confirmed by physical exam
and complementary studies.

REFERENCES
1. Denning DW, Cadranel J, Beigelman-Aubry C, et al. Chronic
pulmonary aspergillosis: rationale and clinical guidelines fordiagnosis and management. Eur Respir J., 2016; 47: 45.
2. Judson MA, Stevens DA. The treatment of pulmonary
aspergilloma. Curr Opin Investig Drugs 2001; 2: 1375.
3. Light RW. Pleural diseases. 6th ed. Philadelphia: Lippincott
Williams & Wilkins, 2013.
4. Rajaratnam S, Bhatt A, Chase S, George O. An unusual cause of
acquired cardiac dextroposition. Curr Med Issues 2017; 15: 237-9.
5. Pernkopf, E, Zeit F. A'nat. und Entwicklungsgeschichte, 1926; 79:579-752.
6. Dev D, Basran GS. Pleural effusion: a clinical review. Monaldi Arch
Chest Dis. 1994;49(1):25-35.
7. Franken EA Jr. Pneumomediastinum in newborn with associateddextroposition of the heart. Am J Roentgenol radium Ther Nucl
Med., 1970; 109(2):252-60.
8. Abdullah MM, Lacro RV, Smallhorn J, et al. Foetal cardiac
dextroposition in absence of an intrathorasic mass: sign ofsignificant right lung hypoplasia. J Ultrasound Med., 2000;19(10):669-76.
9. Hollendonner WJ, Pastor BH. Dextroposition of the heart
simulating congenital dextrocardia. Am J Med., 1956; 20:647-50.
10. Haththotuwa HR, Dubrey SW. A heart on the right can be more
complex than it first appears. BMJ Case Rep 2013; 2013.

Fiziologia - Physiology • 2019.29.1(97)REVĂRSAT PLEURAL TUBERCULOS SAUDEXTROPOZIȚIA INIMII? RAPORT DE CAZ
REZUMATRevărsatele pleurale sunt patologii frecvent întâlnite în practica clinică. Dextropoziția inimii reprezintă poziția orizontală ainimii în hemitoracele drept. Prezentul caz clinic prezintă o dextropoziție congenitală nedecelată, la o pacientă, în vârstă de
61 de ani, fără antecedente cunoscute de patologie pulmonară sau cardiovasculară, internată cu suspiciunea de pleurezie
tuberculoasă. Radiografia toracică, incidență postero – anterioară, a arătat prezența unei opacități omogene care ocupă
jumătatea inferioară a hemitoracelui stâng, o imagine de hipertransparență circumscrisă de un inel opac, situat lângă hilul
stâng, cu deplasarea mediastinului de partea opusă.
Cuvinte cheie: revărsat pleural tuberculos, dextropoziția inimii, radiografie toracică.

Fiziologia - Physiology • 2019.29.1(97)29THE ROLE OF VITAMIN D IN THE PATHOGENESISOF MULTIPLE SCLEROSISALBU VALERIA CARMEN1, SANDU RALUCA ELENA2, BUMBEA ANA-MARIA3,
TARTEA ANCA-ELENA4, BOGDAN CATALIN5, FLORESCU ANDREEA OANA6,
BURADA EMILIA5, TARTEA GEORGICA COSTINEL5, MALIN RAMONA7,BARBULESCU ANDREEA LILI8
1Departament of Neurology, University of Medicine and Pharmacy of Craiova, Romania

2Departament of Biochemistry, University of Medicine and Pharmacy of Craiova, Romania
3Department of Medical Rehabilitation, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania
4 Neuropsychiatry Hospital of Craiova, Romania

5Department of Functional Sciences, University of Medicine and Pharmacy of Craiova, Romania
6Departament of Radiology, University of Medicine and Pharmacy of Craiova, Romania
7Department of Otolaryngology, University of Medicine and Pharmacy of Craiova

8Departament of Pharmacology, University of Medicine and Pharmacy of Craiova, Romania

ABSTRACTMultiple sclerosis (MS) is an autoimmune, inflammatory, chronic, demyelinating disorder of CNS, characterized
by episodes of inflammation and focal demyelination with multiple localizations, disseminated in time, but also
through a process of axonal degeneration that unfolds in parallel. The disease usually has an evolution withflares and remissions, but can also have a constant character of progression over time, which leads toaccumulation of varied neurological deficits and different degrees of disabilities. It is considered to be the mostfrequent affection of the young adult after trauma, the onset age being between 20-40 years with a maximum
at the age of 30 and is 2-3 times more frequent in women than in men, there is no scientific evidence to explain
the increased incidence of the disease in females, the possible explanation being that women are generally
more susceptible to inflammatory and immunological disorders. Although clinical studies on the administrationand effects of vitamin D in various pathologies are limited, experimental and epidemiological data aresufficiently consistent and encouraging, reinforcing the hypothesis that hypovitaminosis D may be a risk factor
for the onset of disease and, at the same time, a predictive factor for the development of multiple sclerosis.
Although the doses of vitamin D used in clinical trials are quite different and vary between 100 IU/day and14,000 IU/day, and the results were encouraging for all groups of patients under study, the administered doses
should be determined for each subject in part taking into account a number of factors such as age, gender,
associated comorbidities, environmental or genetic factors, the latter being able to interact and combine tocause disease. If we consider that this vitamin is involved in a large number of disease, including autoimmune
disease and especially multiple sclerosis, significantly influencing the activity of regulating T lymphocytes,
whose role is well known in the pathogenesis of the disease, it can be concluded that hypovitaminosis D
becomes a major health problem and vitamin D treatment will become a simple and effective therapeutic option
for prospective patients with multiple sclerosis which is known as the young adults disease, a disabling disease
with strong negative impact on their quality of life. Therefore, recommending the administration of vitamin D to
multiple sclerosis patients can be regarded as a wise, provocative and a sufficiently effective option, which iscurrently provisory, to achieve improvement health status in patients with this disease.
Keywords: Multiple sclerosis, autoimmune, inflammatory, chronic, demyelinating disorder of CNS, vitamin D.

Received April 13th 2019; Accepted May 29th 2019. Address for correspondence: Sandu Raluca Elena, Lecturer, MD, PhD, Department ofBiochemistry, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania; phone +40351443 500; email: ralucasandu80@gmail.com

30

Fiziologia - Physiology • 2019.29.1(97)INTRODUCTION
Multiple sclerosis (MS) is an autoimmune, inflammatory,
chronic, demyelinating disorder of CNS, characterized byepisodes of inflammation and focal demyelination withmultiple localizations, disseminated in time, but also througha process of axonal degeneration that unfolds in parallel.
The disease usually has an evolution with flares andremissions, but can also have a constant character ofprogression over time, which leads to accumulation of varied
neurological deficits and different degrees of disabilities. [1].
It is considered to be the most frequent affection of theyoung adult after trauma, the onset age being between 2040 years with a maximum at the age of 30 and is 2-3 times
more frequent in women than in men [2,12], there is no
scientific evidence to explain the increased incidence of thedisease in females, the possible explanation being thatwomen are generally more susceptible to inflammatory andimmunological disorders [1]. Multiple sclerosis has aprevalence of less than 1 per 100,000 inhabitants in theequatorial regions, 6 to 14 per 100,000 inhabitants in the US
and southern Europe, 30 to 80 per 100,000 inhabitants in
Canada, northern Europe and the northern US. The risk ofdeveloping the disease, according to some studies grows
with the increase in latitudes, a fact confirmed by
epidemiological research, so it is considered that AfricanAmericans are at risk compared with the white population
but both races present the same south-north risk gradient.
These studies bring into question the fact that theenvironmental factor has an important role in thepathogenesis of Multiple Sclerosis, regardless of the genetic
predisposition of the individual.(1) Also, there are
epidemiological data indicating the existence of a directcausal relationship in the general population between
latitude, exposure to solar radiation, vitamin D levels and therisk of Multiple Sclerosis as well as immunological data thatshowed vitamin D influences the activity of regulatory T
lymphocytes, lymphocytes with important role in thepathogenesis of the disease [5].
As a result of the dissemination and random localizationof lesions in the central nervous system, clinicalmanifestations can vary: retrobulbar optic neuritis, motor
deficits with varied distribution (most common paraparesis),other signs of pyramid damage, sensitivity disorders in theform of paresthesia (most likely) or Lhermitte sign, impaired
occulomotori nerves (internuclear ophthalmoplegia, thenerve paralysis of occulomotor III, IV, VI with diplopia),
paralysis of the facial nerve, cerebelous syndrome,
vestibular syndrome in particular vertigo, sphincter disorders(retention or urinary incontinence, compelling urge,
constipation), fatigue, depression, cognitive impairment,
sexual dysfunction [1,2,8,12].
Thus, depending on the evolution of the neurologicalsigns and syndromes, four clinical forms are distinguished:
1. Relapsing- remitting MS (RRMS) represents the mostcommon form of disease (60-70% of cases), being the
Fiziologia - Physiology • 2019.29.1(97)apanage of the young age (30years) and ischaracterized by the appearance of the relapses,
clinically reliable; the attack installs quickly (hours ordays), the symptomatology can be submitted
completely or incomplete, and there are no elements
of progression of the disease between the attacks.
Between the relapses there may be time intervals ofmonths or even years [1,2,8,12].
2. Secondary Progressive MS occurs at a time when, inpatients with RRMS, neurological deficits considered
as relapses, are no longer resolved, thus occurring acontinuous progression of the disease interrupted ornot, sometimes with phases in flats. It is believed thata RRMS can turn into MSSP after approximately 10
years of evolution, each patient presenting a risk ofapproximately 2% each year to develop a MSSP
[2,8,12].
3. The primary progressive MS form represents
approximately 15% of all patients, being more frequent
among patients over 40 years of age and characterized
by progressive worsening of neurological deficits fromtheir onset, with no remittance, possibly minor andtemporary improvement, or very rarely, with periods inthe flat, practically cannot describe the clinical attacks.
According to demyelinating lesions localization, thistype of disease affects mainly the spinal cord, fromonset, rather than cerebral level [2, 8,12].
4. Progressive relapsing multiple sclerosis form, the rare
form of illness, characterized by the existence of
attacks, but which is not remitted from the beginningand has, from the onset, a continuous progressive
evolution of the symptomatology. It can be considered
as a particular form of evolution of SMPP or SMSP,
being present in only about 5% of patients [2,8,12].
Although the disease pathogenesis remains somehow
unknown because no etiological factor has been identified,
it is considered that the epidemiological factor should matterand be included in these theories on the etiopathogenesisof the disease. Thus, multiple sclerosis, also known as
plaque sclerosis is considered as having a multifactorial
etiology, being the result of interaction between several
factors: environmental, especially nutritional, vitamin D,
geographic, infectious - possible infections with Epstein-Barrvirus or genetic factors, giving the disease heterogeneous
appearance regarding to clinical, histopathological andgenetic aspects [3,5,6]. Other risk factors possibly involvedin triggering multiple sclerosis are: age, gender, family
history, race, smoking, sun exposure. Smoking increases
the risk of illness, worsens neurological deficits andenhances handicap degree [3,4,6]. It therefore appears thatsome of these factors, such as infections, sun exposure andvitamin D levels, are potential risk factors, especially for theunder-15 population [9].
Nutritional factors play an important role in theoccurrence of the disease, so it is believed that a diet high31

in animal fats increases the risk of developing MS as a resultof the increase in cell membrane rigidity, the cell becoming
vulnerable to various factors, and a diet rich in oils reduces
membrane rigidity maintaining structural integrity of the cell.
So, studies conducted in Asia, where the consumption ofoils is superior to saturated fat, have shown an important
decrease in the incidence of this disease, and a diet rich inpolysaturated fatty acids and vitamin D decreased multiple
sclerosis prevalence according to studies conducted by agroup of Norwegian fishermen [10].
It has been reiterated the idea that hipovitaminosis D, as
well as sun exposure, combined, or interacting with otherenvironmental or genetic factors, represent risk factors for
multiple sclerosis. It is known that skin exposure toultraviolet radiation (UVB) is essential for the vitamin D
biosynthesis, which is the major source of vitamin D for mostindividuals, and therefore sun exposure for short periods inthe case of people living in areas over 40 degrees northernlatitude or in temperate regions where the amount of
ultraviolet radiation is low or in areas with short days andlong nights longer than 6-8 months / year, the synthesis ofvitamin D decreases [4,9,15]. Also, a diet rich in fish fat is an
important source of exogenous vitamin D. [15]. There are
two forms of vitamin D: vitamin D2 (ergocalciferol) that is
produced by some plants and vitamin D3 (colecalciferol)synthesized in the skin of humans and animals, which is themost important source of vitamin D exogenous, the mostactive biological form (9). Vitamin D exerts a number offunctions in the human body acting through the vitamin D
receptors present in most human body tissues, apparently
playing an immunomodulatory role in CNS. From this pointof view, vitamin D levels, and especially hypovitaminosis D,
present an increasing interest in the production andtriggering of the disease, and the correction of thisdeficiency may become a new effective therapeutic target
for multiple sclerosis [6,13,14].
Vitamin D is a steroidal hormone successively
metabolized by UVB radiation in the skin, liver under theaction of several vitamin D-25-hydroxylase enzymes, themost important of which is CYP2R1 and kidney in theproximal tube under the action of the 1-alpha hydroxylase
enzyme (CYP27B1 ) to the active metabolite 1,25
dihydroxyvitamin D (CALCITRIOL), which in turn acts on
tissues containing certain vitamin D receptors such as skin,muscle, bone, gonads, intestinal, CNS, microglia,
macrophages, dendritic cells, activated monocytes and B
and T lymphocytes, having a role in modifying transcription,in proliferation and differentiation of immune cells [4,15].
Therefore, hypovitaminosis D is one of the most studied
environmental risk factors capable of producing MS, and thecorrection of this deficiency can become an important link intreating this disease [10]. Exogenous or produced vitamin D
in the skin is transformed in the liver until 25-hydroxyvitamin
D (25-OH-VITAMINE or COLECALCIFEROL) which
represents its major circulating form and its plasma
concentrations are influenced by vitamin D intake and32

exposure to sunlight, this metabolite being actually the one
determined in the lab [4,15].
It is known that the fundamental role of this vitamin is tokeep bone structures intact, to interfere with the metabolism
of calcium and phosphorus, but it also exerts other generaleffects such as: acting in the regulation of cardiovascularsystem activity, anti-inflammatory, anti-proliferative effects(for certain types of cancer), modulators of neurotrophins,
growth factors and neurotransmitters from mammalian CNS,
immunomodulators, the latter being involved in theoccurrence of autoimmune diseases such as type 1
diabetes, Chron disease, rheumatoid arthritis, and last but
not least, multiple sclerosis [4,6,15].
The 25-OH-vitamin D plasma concentration of 80 nmol/l(30 ng/ml) is considered to be necessary both for maximum
intestinal absorption of calcium and for the delivery of
extra1-alpha-hydroxylase, which is present in more tissues,
where it produces 1,25 dihydroxyvitamin D [15]. Because,
following experimental and immunological studies, it has
been found that vitamin D has a similar mode of action tointerferon beta, it is assumed that it can have an important
immunomodulatory effect [7,15]. Although it is known thatthe population of the northern countries is deficiency invitamin D, there are studies which have shown that thisdeficiency may be present even more than was believed insunny countries such as India and Australia [15]. There is a
study, considered as a reference that looked at the risk ofdeveloping MS depending on the serum level of 25-OHvitamin D in healthy individuals, meaning no clinical signs
suggestive of MS. It was concluded that subjects with a highlevel of vitamin D, i.e. those with 99-152 nmol/L, had asignificantly lower risk of developing the disease than those
who had a low vitamin D level, ie between 15-63 nmol/L.
Therefore, it appears that the risk of developing MS is lower
provided that the serum level of 25-OH-vitamin D is
maintained at levels above 100 nmol/l in children oradolescents. (11)A small number of clinical trials have been performedthat have enrolled patients with multiple sclerosis remitting
remission, in which 25-OH- vitamin D dosages have been
performed and which have shown an apparent number ofpatients, over 83% of them had serum levels of 25-OHvitamin D below 75 nmol/l, and 17% of patients had 25-OHvitamin D deficiency, i.e. below 25 nmol/l. Also, more than
95% of the patients analyzed never reached the serum levelof 100 nmol/l of 25-OH-vitamin D. Thus, it can be concluded
that most multiple sclerosis patients have low serum vitaminD levels, and it has been estimated that nearly threequarters of multiple sclerosis cases could be avoided if
vitamin D plasma levels were above 100nmol / L(10). In the
study conducted by Mahon and colleagues who followed
multiple sclerosis patients treated with 1000 IU/day ofvitamin D for 6 months, it was observed that in fact theconcentrations of (TGF-β1) transforming growth factor beta
1 increased significantly during the course of the study,
without influencing the levels of tumor necrosis factor alphaFiziologia - Physiology • 2019.29.1(97)(TNF-alfa), interferon gamma (IFN-gamma) or interlekin 3(IL-13) after vitamin D treatment. It may be considered that
TGF-β1 serum levels associated with elevated serum levelsof vitamin D may favor the recovery or improvement of thestatus of MS patients. (7; 16) Canadian researchers also
demonstrated that the use of high doses of vitamin D,
approximately 14,000 IU/day, for a period of 6 months - 1
year, do not produce hypercalcemia or notable side effects,
even if the vitamin D serum level is 400nmol/l [15].
Although clinical studies on the administration andeffects of vitamin D in various pathologies are limited,
experimental and epidemiological data are sufficiently
consistent and encouraging, reinforcing the hypothesis thathypovitaminosis D may be a risk factor for the onset of
disease and, at the same time, a predictive factor for thedevelopment of multiple sclerosis. Although the doses ofvitamin D used in clinical trials are quite different and vary
between 100 IU/day and 14,000 IU/day, and the results wereencouraging for all groups of patients under study, theadministered doses should be determined for each subject
in part taking into account a number of factors such as age,gender, associated comorbidities, environmental or genetic
factors, the latter being able to interact and combine tocause disease [15]. If we consider that this vitamin is
involved in a large number of disease, including
autoimmune disease and especially multiple sclerosis,
significantly influencing the activity of regulating T
lymphocytes, whose role is well known in the pathogenesisof the disease, it can be concluded that hypovitaminosis D
becomes a major health problem and vitamin D treatment
will become a simple and effective therapeutic option forprospective patients with multiple sclerosis which is known
as the young adults disease, a disabling disease with strong
negative impact on their quality of life [5].
Therefore, recommending the administration of vitamin
D to multiple sclerosis patients can be regarded as a wise,
provocative and a sufficiently effective option, which iscurrently provisory, to achieve improvement health status inpatients with this disease.
AUTHOR CONTRIBUTION
The authors contributed equally to the manuscript and share firstauthorship.

Fiziologia - Physiology • 2019.29.1(97)

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33

ROLUL VITAMINEI D ÎN PATOGENIA
SCLEROZEI MULTIPLE
REZUMAT
Scleroza multiplă (S.M.) este o afecţiune autoimună, inflamatorie, cronică, demielinizantă a SNC, caracterizată prinepisoade de inflamaţie şi demielinizare focală cu localizări multiple, diseminate în timp, dar şi printr-un proces dedegenerescenţă axonală care se desfasoară în paralel. Boala are de obicei, o evoluţie cu pusee şi remisiuni, dar poate
avea şi un caracter constant de progresivitate în timp, ceea ce conduce la acumulare de deficite neurologice variate şi ladizabilităţi de grade diferite. Deşi studiile clinice în ceea ce priveşte administrarea şi efectele vitaminei D în diverse patologii
sunt limitate, totuşi datele experimentale şi epidemiologice sunt suficient de consistente şi în acelaşi timp încurajatoareîntărind ipoteza că hipovitaminoza D poate fi un factor de risc pentru declanşarea bolii şi în acelaşi timp un factor predictiv
de evoluţie al sclerozei multiple. Cu toate că dozele de vitamina D folosite în studiile clinice sunt destul de variate şi diferiteîntre 100 UI/zi până 14.000 UI/zi, iar rezultatele au fost încurajatoare pentru toate loturile de pacienţi luaţi în studiu, dozele
administrate ar trebui stabilite pentru fiecare subiect în parte tinând cont de o serie de factori precum vârsta, sexul,
comorbidităţile asociate, factorii de mediu sau genetici, aceştia din urmă putând interacţiona şi combina astfel încât să
determine boala. Dacă avem în vedere faptul că această vitamină este implicată în apariţia unui numar destul de mare deafectiuni, inclusiv bolile autoimune şi în special scleroza multiplă , influentând semnificativ activitatea limfocitelor T
reglatoare, al căror rol este bine cunoscut în patogenia bolii, se poate afirma că hipovitaminoza D devine o problemă majoră
de sănătate, iar tratamentul cu vitamina D va deveni o opţiune terapeutică simplă şi eficientă, de perspectivă, pentru
pacienţii cu scleroză multiplă care este cunoscută ca fiind boala adultului tânăr, o boală dizabilitanta şi cu un puternic impact
negativ asupra calitătii vieţii acestora. De aceea, recomandarea administrării de vitamina D la bolnavii cu SM poate fi privită
ca o opţiune înţeleaptă, provocatoare şi suficient de eficientă, momentan provizorie, care sa aibă ca rezultat îmbunătăţirea
stării de sănătate a pacienţilor cu această boală.
Cuvinte cheie: Scleroza multiplă, autoimună, inflamatorie, demielinizantă a SNC, vitamina D.

34

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