2021.31.1 (101 )  Fiziologia - Physiology

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Fiziologia - Physiology  2021.31.1 (101)CONTENTS
Fiziologia-Physiology 2021.31.1(101)
1. Exosomes as valuable players in health and diseases
Alexandru Tîrziu, Dana Plesca, Simona Anghel, Carmen Panaitescu, Virgil Păunescu
2. Physical agents as antimicrobial factors
Carmen Tatu, Gabriela Tănasie, Alina-Georgiana Simina, Cristina Gaspar, Marius Georgescu, Florina Bojin, Daniela
Puscasiu, Carmen Panaitescu
3. Assessment of heart rate changes to posture and orthostatic tolerance in a cohort of male nigerian
undergraduates
Olusoji A. Adalumo, Saheed A. Lawal, Funmilayo M. Owolabi
4. Exploration of the association between classical and emerging risk factors for diabetic kidney disease andalbuminuria in a cohort of type 2 diabetes mellitus patients
Marcel Stoită, Cosmin Mihai Vesa, Amorin Popa
5. Necrotizing fasciitis in a patient with hepatic encephalopathy. Case report and literature review
Diana Maria Chiorean, Monica Daniela Cotarcă, Beatrice Barac, Raluca Niculescu, Mihaela Cornelia Șincu, AndreeaCătălina Tinca, Iuliu Gabriel Cocuz, Adrian Horatiu Sabău, Paul Antoniu Botan, Ovidiu Simion Cotoi
6. The genetic and acquired host susceptibility to non-tuberculous mycobacterial infections
Biciusca Viorel, Sorina Ionelia Stan, Patricia Durand, Mara Balteanu, Biciusca Teodora, Beatrice Mahler
CUPRINS
1. Rolul exozomilor în procesele fiziologice și patologice umane
Alexandru Tîrziu, Dana Plesca, Simona Anghel, Carmen Panaitescu, Virgil Păunescu
2. Agenții fizici ca factori antimicrobieni
Carmen Tatu, Gabriela Tănasie, Alina-Georgiana Simina, Cristina Gaspar, Marius Georgescu, Florina Bojin, Daniela
Puscasiu, Carmen Panaitescu
3. Evaluarea modificărilor ritmului cardiac la postură și toleranță ortostatică la o cohortă de studenți nigerieni
de sex masculin
Olusoji A. Adalumo, Saheed A. Lawal, Funmilayo M. Owolabi
4. Explorarea asocierii între factorii de risc cunoscuți și emergenți pentru boala renala diabetică și respectiv
albuminurie la pacienții cu diabet zaharat de tip 2
Marcel Stoită, Cosmin Mihai Vesa, Amorin Popa
5. Fasceita necrotizantă la un pacient cu encefalopatie hepatică. Prezentare de caz și analiza literaturii despecialitate
Diana Maria Chiorean, Monica Daniela Cotarcă, Beatrice Barac, Raluca Niculescu, Mihaela Cornelia Șincu, AndreeaCătălina Tinca, Iuliu Gabriel Cocuz, Adrian Horatiu Sabău, Paul Antoniu Botan, Ovidiu Simion Cotoi
6. Susceptibilitatea genetică și dobândită a gazdei la infecțiile micobacteriene non-tuberculoase
Biciusca Viorel, Sorina Ionelia Stan, Patricia Durand, Mara Balteanu, Biciusca Teodora, Beatrice Mahler
2021.31.1 (101 )  Fiziologia - Physiology

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EXOSOMES AS VALUABLE PLAYERS IN HEALTH AND
DISEASESALEXANDRU TÎRZIU1, DANA PLESCA1,2, SIMONA ANGHEL1,2, CARMEN PANAITESCU1,2,
VIRGIL PĂUNESCU1,2
Department of Functional Sciences, „Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
OncoGen-Centre for Gene and Cellular Therapies in the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency County Hospital Timisoara, Romania

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ABSTRACT
Normal human physiology involves a well-coordinated cross-talk between different cell types. Throughout evolution, multicellular organisms
have developed a variety of signalling pathways in the local microenvironment, as well as distant sites. Besides hormones and proteinprotein interactions, exosomes provide an alternative communication strategy with high specificity and stability and increased penetrability
in various organs and tissues due to their low immunogenicity profile and reduced size.

In this review we will focus on exosome biogenesis, their role in coordinating various physiological and pathologicalpathways, different strategies of exosomal isolation, as well as potential future applications in early diagnostic
and drug delivery.
Key words: exosome, inflammation, metastasis, angiogenesis, cell differentiation, tumor microenvironment

INTRODUCTION
Microvesicles are small, lipid-enclosed vesicles that are
released into the extracellular space to facilitate intercellularcommunication. Apart from apoptotic bodies, ectosomes andendosomes exert pleiotropic effects, being involved in complex
processes such as angiogenesis, cell differentiation, adhesion
and inflammation [1-3].
Microvesicles transport various biomolecules such as lipids,
proteins or nucleic acids. Exosomal transmembrane and surface
proteins facilitate protein-protein interactions from distant cells.
Specific immune response relies on antigen-presenting cellderived exosomes containing MHC class I and class II molecules,
which aid in early pathogen recognition [4-5].
Immunosuppressive and angiogenic exosomes express
PD-L1 or VEGF, favoring the formation of a hospitable tumor
microenvironment before the arrival of cancer cells in the process
of metastasis [6].
Nucleic acids found in the exosomal cargo include
microRNAs (miRNAs) and messenger RNAs (mRNAs) whichalter the expression of target genes and hence, modulate cellularsignaling pathways involved in growth, differentiation, adhesion
and migration [7].
Similar to liposomes, specific cell-derived exosomes canbe loaded with drugs in order to improve their pharmacokinetics.
However, drug loading techniques involve a sufficient number of
exosomes with intact biological functions [8]. A large and variate

palette of isolation techniques exists in the actual research fieldand each technique has its own advantages and disadvantages[9]. Further research is required to improve the yield of exosome
isolation and drug internalization. Finally, drugs that were once
discarded due to their poor pharmacological properties can bereconsidered by encapsulating them into extracellular vesicles.

MICROVESICLES NOMENCLATURE
Microvesicles released in the extracellular milieu are
divided into three categories based on their size and cellular
activity: exosomes, shedding microvesicles and apoptotic
blebs [1, 10].
Shedding microvesicles (SMVs) or ectosomes are large
vesicles (100-1000 nm) released by plasma membrane budding.
The process of budding is mediated by proteins such as calpain,
flippase, floppase, scramblase and gelsolin [1]. Compared to
exosomes, SMV cargo does not undergo prior selection before
entering the lumen.
Apoptotic blebs are platelet-sized vesicles (approximately
500 nm) released into the extracellular space by cells undergoingprogrammed cell death. ABs are phagocytized by neighboring
cells and their contents are used in further biomoleculesynthesis. Therefore, they are not involved in intercellularcommunication [10].
Exosomes are extracellular vesicles with origin in

Received 12th of May 2021. Accepted 11th of June 2021. Address for correspondence: Alexandru Tîrziu, Department of Functional Sciences, „Victor Babes”
University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Romania; phone/fax: +40256490507; e-mail: alex.tirziu@
gmail.com

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Fiziologia - Physiology  2021.31.1 (101)the endolysosomal compartment [10] and display different
biogenesis, release pathway, size (30-150 nm), content andfunction than SMVs and Abs [7] (Figure 1).

vesicle formation. Members of the tetraspanin family such as
CD9, CD81 or CD63 can be used as markers for exosomal
isolation in immune-affinity assays [10,12].
Membrane curvature and budding is also facilitated bycertain lipids present in the exosomal membrane - ceramides,
cholesterol and extracelullar facing phosphatidyl-serine [6].
Compared to proteins that are secreted in the Golgi
apparatus or in the ESCRT-dependent pathway that require
specific signaling sequences, ESCRT-independent proteins do
not obey known rules. Ras-Carmona et al. developed a randomforest-based algorithm that predicts proteins that are more likely
to be included in exosomes, allowing further biomarker discovery[13] (Figure 2).

Fig. 1. Shedding microvesicles are released by the outward buddingof the plasma membrane. Exosomes are produced by inwardbudding of the multivesicular body and subsequent fusion with theplasma membrane. Apoptotic blebs are released when cells undergo
programmed cell death and contain condensed nuclear material anddegraded organelles.

BIOGENESIS OF EXOSOMES
Exosomal production starts with the process of plasma
membrane invagination forming an early endosome. The
endosomal membrane suffers an inward budding (into thelumen), creating membrane-bound vesicles in a structure called
multivesicular body (MVB). The vesicles are then separated
from the membrane in a twisting motion resulting intraluminal
vesicles (ILVs). The last step involves the fusion of the plasma
membrane with the MVBs and the release of ILVs into theextracellular space. The zipper-like fusion process is mediatedby the interaction between v-SNARE (present on the MVB) andt-SNARE (the target plasma membrane) forming the SNARE
complex [11].
The classical pathway for exosome formation depends
on the ESCRT (endosomal sorting complexes required fortransport) multimeric protein complex. ESCRT comprises 4
protein complexes with specific, well-coordinated functions.
ESCRT0 recruits the proteins that will be translocated into theintraluminal vesicle. In the classical pathway, ubiquitinylated
proteins enter in the intraluminal space [1]. ESCRT-1 and
ESCRT-2 are the main effectors of membrane budding and
ILV formation in the intracellular compartment, while ESCRT-3
and the VPS4 ATP-ase cleave the membrane, releasing themembrane-bound vesicle into the intraluminal space. ESCRTindependent exosome biogenesis depends on the tetraspanin
protein family which induces membrane curvature and allows
2021.31.1 (101 )  Fiziologia - Physiology

Fig. 2. Exosome biogenesis starts with plasma invagination (top left)
and creates an early endosome. In the ESCRT-pathway, the ESCRT
protein complex mediates the intraluminal vesicle formation (ILV) andprotein and nucleic acid enrichment inside the ILVs. The multivesicularbody (MVB) fuses with the plasma membrane in a SNARE-mediatedfashion, releasing the exosomes in the extracellular space. The ESCRTindependent release involves ceramide produced by sphingomyelinase(SM-ase) or tetraspanins (CD9, CD81, CD63) to induce the membrane
curvature.

Exosomes influence the target cells in four ways. The
first mechanism involves the direct protein-protein interaction
between the exosomal proteins and the target cell. In the second
mechanism, the surface proteins are cleaved by extracellular
proteases, exposing different binding sites that can interact
with the target cell. Direct fusion of the cell membrane with theexosomal particle facilitates miRNA and mRNA transfer inside
the cytoplasm. The last pathway consists of internalization of theexosome via endocytosis or phagocytosis [10].

BIOLOGICAL FUNCTIONS OF EXOSOMES
Antigen presentation and lymphatic trafficking
Srinivasan et al. showed that exosomes containing foreign
antigens are rapidly (in a matter of minutes) transported from5the infection site into the lymphatic system. Regional lymphatic
vessels express high permeability for exosomal particles
facilitating translocation into the lymphatic vessel lumen.
Extracellular vesicles released during the inflammatory
response originate from resident antigen-presenting cells [10]and from normal infected cells. Exosome release in the lymphatic
stream allows rapid flow of information that precedes the arrivalof effector cells at the infection site [5].
Once reaching the lymph node, the exosomes are uptakenby B-cells and macrophages which prime the helper and cytotoxic
T cells resulting in a prompt and specific immune response.
T-cell-derived exosomes express TCR which in turn trigger
interferon-gamma and granzyme production in naive CD8+ T
cells, leading to an inflammatory positive feedback [7].
This phenomenon was demonstrated in mice infectedwith M. tuberculosis that expressed high levels of infected
macrophages-exosomes containing mycobacterial lipoproteins[4].
The lymph node route is also exploited by melanomas,
tumors characterized by initial lymphatic dissemination. Once inthe lymphatic system, melanoma-derived exosomes induce animmunosuppressive environment allowing subsequent lymphatic
invasion. Recent work showed that B16F10 melanoma cellderived exosomes reached the subcapsular sinus macrophages
and inhibited their anti-tumor functions [6].
Immune regulation and pregnancy
Pregnancy involves a tightly regulated bilateralcommunication between the fetal and maternal organisms
mediated by hormones, cytokines and exosomes which involve
fetal vasculogenesis. Dysregulation in exosome composition
or number are associated with gestational-related pathologies
such as pre-eclampsia, gestation diabetes or fetal growth
restriction [14].
Exosomes produced by fetal cells interact with specificmaternal cells to induce maternal changes required to sustain
fetal growth. Placental-derived extracellular vesicles express
immunomodulatory molecules such as PD-L1 or CD276 thatprevent an undesired cellular immune response against the fetus.
MiRNAs miR-516b-5p, miR-517-5p, and miR-518a-3p
present in placental-derived exosomes modulate the PI3K-Akt
and the insulin signaling pathways, suggesting their critical rolein gestational diabetes or intra-uterine fetal growth restriction.
Endometrial-derived exosomes contain hsa-miR-30d, a microRNA that enhances integrin subunits α7, β3 and cadherin-5,
facilitating trophoblast invasion and adhesion. Changes in the
spiral artery microarchitecture involve hypertrophy, hyperplasia,
apoptosis and extracellular membrane remodelling, all mediatedby extracellular vesicles released by the cytotrophoblast [15].
Hypoxic conditions stimulate the release of VEGF-containing
extracellular vesicles by the placenta, leading to increased bloodflow and angiogenesis. HUVEC cell line, treated with exosomes
isolated from the plasma of pregnant women stimulated
proliferation, migration and angiogenesis [16].
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Exosomes modulate the tumor microenvironment
architecture and facilitate tumor metastasis
The tumor micro-environment is an intricate biologicalmilieu that involves a complex communication between tumor
and normal resident cells, such as fibroblasts, endothelial cells
or immune cells [17]. By hijacking the physiological anti-tumor
immune response, angiogenesis and wound healing processes,
malignant cells assure their survival, proliferation and invasionin regional or distant sites [18].
Besides cytokine release, cancer cells communicate
through extracellular vesicles that contain various bioactive
molecules. Nucleic acids such as micro-RNAs or small interfering
RNAs can alter the expression of tumor suppressors in target
cells, leading to abnormal cellular proliferation. Proteins involvedin cell signaling such as growth factor receptors can integrate
into the plasma membrane of the target cell, allowing increased
sensitivity to growth factors [1,6].
Recent studies have shown that enzymes involved inadenosine metabolism are highly expressed on the tumorderived exosomal surface. Molecules such as CD73 and CD39
hydrolyze adenosine triphosphate to adenosine in the tumor
microenvironment. High concentrations of adenosine interact
with adenosine receptors A2A and A2B, limiting the proliferation ofcytotoxic T cells [7,19,20]. High expression of CD73 and CD39
is associated with a higher stage malignancy (III/IV) and can beused as a future biomarker to predict prognosis [19].
Tumor-associated exosomes can pass into the bloodstream
and allow cellular communication at distant sites. Signals emitted
by tumor cells favor the development of a pre-metastatic niche,a cluster of different non-tumoral cells present in a target organ
for metastasis (liver, lung, bone etc.), that creates a hospitable,
immunosuppressive, highly vascularized microenvironment. By
creating the pre-metastatic niche, the tumor educates the target
organ how to prepare for its arrival.
Pre-metastatic niche formation is a well-coordinated multistep phenomenon that involves several crucial steps. Tumor
growth, invasion and metastasis are highly dependent on the
nutrients provided by blood flow. Pro-angiogenic extracellular
vesicles contain high concentrations of cytokines such as VEGF
(vascular endothelial growth factor) or long-noncoding RNAs
such as aHIF (antisense hypoxia-inducible factor) [21]. Tumorderived proangiogenic vesicles can also function as a cellulardisposal mechanism, contributing to drug resistance in targetedcancer therapy with VEGF inhibitors [10,21].
Local invasion and angiogenesis rely on extracellular
membrane remodelling. Pancreatic cancer cells release in the
portal circulation exosomes loaded with macrophage migration
inhibitory factor (MIF). MIF induces TGF-β secretion from Kupffer
cells, which in turn stimulates fibronectin production by stellate
cells in the perisinusoidal spaces of the liver [6]. Peritoneal
metastasis is promoted by exosomes containing MMP-1 (matrix
metalloproteinase 1) secreted by ovarian cancer cells in mice
[22].
The immunosuppressive tumor microenvironment relies
upon the secretion of exosomes. Tumor-derived exosomes
Fiziologia - Physiology  2021.31.1 (101)induce differentiation of monocyte and M1 macrophages
to an anti-inflammatory phenotype. TGF-β, IL-10 and othercytokines reduce the local activity of anti-tumoral T-lymphocytes.
Additionally, several subpopulations of exosomes express
the pro-apoptotic molecules FasL and PD-L 1. Cytotoxic T
cell response is also hampered by the increasing number ofregulatory T cells and BMDCs (bone-marrow derived dendritic
cells).
Tumor metastasis organotropism depends on the adhesion
molecules expression pattern on exosomes. Tumor-derived
exosomal cargo expresses different integrin patterns which
decide the organs in which the pre-metastatic niche forms andmetastasis occurs. Relevant examples include α6β4 and α6β1
integrins in breast cancer with pulmonary metastasis or αvβ5
integrin enriched exosomes in hepatic metastases of pancreatic
adenocarcinomas. The integrin patterns on TEVs (tumor
extracellular vesicles) are highly organ-specific and may predict
future organ metastasis in cancer patients [6].
Endometriosis
Endometriosis is a chronic inflammatory diseasecharacterized by the presence of ectopic hormone-dependentendometrial-like tissue containing glands and stoma, leading
to pain and infertility. Freger et al. demonstrated that miRNAs,
lncRNAs and proteins contained within the exosomes contributeto the characteristic cell signaling malfunctions – angiogenesis,
chronic inflammation, invasion, migration, proliferation andapoptosis [14].
Greening et al. showed that endometriosis cell-derived
exosomes induce fundamental changes in normal trophoblastic
cells involving adhesion capacity by modulating the FAK (focal
adhesion kinase) pathway [23].
Endometriosis involves a complex cross-talk between
stromal-ectopic cells and M2-macrophages. The presence ofectopic endometrial tissue in target organs (ovary, peritoneum,utero-sacral ligaments) modulates the differentiation of circulating
monocytes to a M2 phenotype with subsequent release of
immunosuppressive cytokines (IL-10, TGF-β) that allows ectopic
tissue implantation [14].
One mechanism proposed for extrauterine endometrial
invasion involves retrograde menstrual cell flow into the pelvic
area. Along with menstrual cells, exosomes that pass in a
retrograde fashion can prime the ectopic situs for implantation
by enhancing angiogenesis. Endometriosis lesions express highvascularization, dependent on the menstrual cycle [24].
Another mechanism that can explain endometriosis
developed in women with Rokitansky-Mayer-Kuster-Haster
disease involves coelomic metaplasia, a process in which cells
that line the peritoneal or pleural cavities transdifferentiate
into functional endometrium. The process of metaplasia might
involve miRNAs, mRNAs or regulatory proteins present in the
exosomal cargo.
The angiogenic-related lncRNA aHIF (antisense
hypoxia-inducible factor) was found in high concentrations in
2021.31.1 (101 )  Fiziologia - Physiology

ectopic endometrial cells as well as endometriosis patients’sera. Supporting evidence show that HUVEC treated withendometriosis-derived exosomes showed high VEGF releaseand b-FGF, suggesting enhanced angiogenesis [25].
Diabetes and cardiovascular diseases
Diabetes mellitus represents an important risk factor for
cardiovascular disease development due to the pro-inflammatory
and hormonal imbalance that leads to atheroma plaqueformation. Extracellular vesicles identified in the sera of DM
patients provide additional information about the interconnection
the role of DM in atherosclerosis biology.
The main actors involved in atherosclerosis are theendothelial cells, vascular smooth muscle cells and monocytes/
macrophages. Endothelial dysfunction stimulates the releaseof IL-6 and IL-8-containing extracellular vesicles that promote
monocyte migration to the subintimal layer. Extracellular vesicles
also promote cell adhesion by enhancing expression of ICAM-1
in endothelial cells, as well as CD11b in monocytes. Moreover,microRNAs such as miR-10-a promote NF-KB signaling pathway
in monocytes leading to a local pro-inflammatory state [10].
Vascular homeostasis implies the existence of a crosstalk between the endothelial cells and the vascular smoothmuscle cells. Shear stress that acts on the endothelial surface
provides a stimulus for changes in vascular tone [26]. Heo et
al. demonstrated that in arterial injury, exosomes released byendothelial cells and platelets contain PDGF (platelet-derived
growth factor) which induces phenotypic changes in vascularsmooth muscle cells – from a contractile phenotype to a collagensecreting one. Moreover, the miRNA repertoire of VSMCs
altered the endothelial biology, enhancing EC proliferation andmigration [27].
Adipocyte-derived exosomes from obese patients act
in an endocrine fashion on macrophages, potentiating theirinflammatory response. Paracrine signaling on neighboring
adipocytes impairs glucose uptake and promotes insulinresistance and altered response to glucose.
The link between obesity, diabetes and atherosclerosis
can be found in the insulin-resistant adipose cell exosomes.
Molecules such as sonic the hedgehog protein enhance theexpression of pro-inflammatory cytokines (TNF-alpha, IL-1beta,IL-6) and adhesion molecules (ICAM-1), promote vascularremodeling via MMP2 and MMP9 in the atheroma plaque andpromote vasa vasorum angiogenesis (VEGF-A) [10].
Serum biomarkers used in diabetes (glycemia, glycatedhemoglobin) suffer deviations from the normal values only
in clinically manifest disease. Because of their biomolecularsimilarity with the parental cell, exosomes reflect cellularalterations before the disease onset. Extracellular vesicles can beisolated from any extracellular fluid (blood, urine, cerebrospinal
fluid, peritoneal tap etc.) and can predict the individual risk fordeveloping a certain pathology or complication [1,8].

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EXOSOME ISOLATION AND ANALYSIS PROCEDURES
Ultracentrifugation
Ultracentrifugation is considered the classical method
for isolating exosomes. [6] After a low-speed centrifugation toremove all the cells and debris from the suspension, a variable
number of centrifugation cycles with increasing rotational force
and cycle time will make smaller particles to pellet sequentially.
When reaching 100,000-120,000 x g, exosomes can be isolated
from the suspension.
The disadvantages of this procedure include protein andlipoprotein contamination, vesicle aggregation which leads to alower yield [3] and high mechanical stress due to high centrifugal
forces that may alter exosomal physical and biological properties.
Exosomes isolated using ultracentrifugation alone cannot be
used for drug delivery systems or functional analysis [9].
Gradient-density ultracentrifugation
Derived from blood cell separation techniques, gradientdensity centrifugation exploits the physical property of particles
with a specific density to remain suspended in the fluid region
with the same density.
The density gradient is created using various concentrationsof a biocompatible solution such as dextrose or iodoxinol. The
sample of interest is added to the tube and centrifuged for
16 h at 100,000 x g. After the ultracentrifugation step, all thecomponents migrate to their corresponding density zone andreach isopycnic position.
Compared to ultracentrifugation alone, density-gradientultracentrifugation provides a better separation of extracellular
vesicles from protein aggregates, mainly because the proteinsare denser. Additionally, the density gradient provides a cushion
for mechanical stress, maintaining the EV structure andbiological function intact [3]. However, it cannot isolate strictly the
exosomes, because certain microvesicles with different sizes andcomposition may show an equal density (1.1-1.21 g/ ml) [1,9].
To overcome this problem, a moving-zone density
separation is required. This process involves a solution with adensity significantly lower than of the particles that need to be
separated. After centrifugation, all the suspended particles will
begin to sink proportionally to their mass/size, allowing further
vesicle separation [9].
Size-exclusion chromatography
By passing an aqueous solution through a porous stationary
phase, molecules behave differently based on their size. Smallermolecules tend to pass through the pores and are slowed down by
their longer traffic distance. Larger molecules that cannot fit inside
the pores are forced around the porous particles and released
faster. Therefore, by collecting the particles that pass through thechromatography column at specific time intervals, a higher yield
exosome isolation is obtained.
Because SEC is performed by passive gravity flow, it does notaffect the EVs integrity and preserves their biological functions [9].
8Precipitation
Precipitation utilizes highly hydrophilic polymers, suchas PEG (polyethylene glycol) [12] that interact with the watermolecules that surround the exosomes. PEG-water interactions
create a peri-exosomal hydrophobic micro-environment whichfavors their precipitation.
The drawback of this method is that PEG precipitates not
only exosomes, but also other biomolecules, such as proteinsand nucleic acids. The solution to this inconvenient is an ATPS
(aqueous two-phase system) consisting of a denser, morehydrophilic dextran solution and a PEG solution. After a lowspeed centrifugation cycle, the exosomes migrate in the dextran
phase, while the proteins and nucleic acids migrate in the PEG
phase [9,12].
Immune-based assays
Monoclonal antibodies have high specificity for their target
protein and have been largely used in clinical applications andresearch. Flow cytometry exosome isolation involves antibodies
linked to fluorescent molecules and require a lower detection
threshold due to exosomal significantly reduced diameter.
Immune-based assays provide a high yield exosome
separation with the possibility to isolate certain exosomal
sub-populations [12]. Exosomes express non-specific markers
that aid in basic exosomal isolation. These markers include:
LAMP2B (lyososme associated membrane protein 2B), heatshock proteins, PDGF receptors, flotilin, Rab5, CD81, CD82,
CD63, CD9, and Alix [10].
By detecting markers originating from their parental cells,
exosomes can further be divided into subpopulations. Examples
include EPCAM and EGFR for early cancer detection [1,9].
Immunological assays can only be used for diagnostic
purposes because of their non-neutral pH and non-physiologicalelution buffers (for separating exosomes from the antibodies)that affect irreversibly the biological function of the collected
exosomes [9].
Tim-4-based isolation
In 2016, Nakai et al. developed a high-yield, nonimmunological exosome isolation based on the interaction
between the protein Tim-4 and the phosphatidyl-serine molecules
present on the exosomal surface. This Ca 2+-dependentinteraction can be reversed by adding a calcium chelating agent
such as EDTA (ethylene-diamino tetraacetic acid) allowing
exosome recovery with intact biological function [28].
Microfluidics
Microfluidics-based approaches provide an attractive
alternative to classic exosome separation techniques due to theirhigh yield, low amount of patient sample (several microliters)
and short isolation time (several minutes, compared to the 16
h ultracentrifugation [9,23]), allowing future bed-side diagnostic
tools.
Immunoaffinity-based microfluidics involve passing a
Fiziologia - Physiology  2021.31.1 (101)laminar flow of sample into chip channels coated with exosomespecific antibodies (anti-CD63, CD9, CD81). To increase theprobability that the antibodies will bind to their specific target, alarger surface binding area is required [9].
Zhang et al. developed a three-dimensional Y-shaped
lattice that increased the surface area, allowing better antigen
binding. The isolation interface was built using graphene oxide,
polydopamine and streptococcal protein G that interacts with the
Fc portion of the antibodies used for exosomal selection [29].
Size-based microfluidic separation separate exosomesfrom other cellular and molecular components based on theircharacteristic size (30-150 nm). Exosome Total Isolation
Chip (ExoTIC) utilizes a series of nano-filters with decreasing
pore size so that particles smaller than exosomes are filteredout, leading to a concentrated exosome suspension. ExoTIC
effectively separates exosomes (4-1000 times better thanultracentrifugation) from cell culture media as well as some bodily
fluids (urine, bronchoalveolar lavage) [30].
Wang et al. created a chip architecture consisting of ciliatedmicropillars (micropillars with silicone nanowires). The space
between the silicone nanowires is equal to the exosome diameter,
allowing exosome entrapment between the nanowires andpassage of free proteins and nucleic acids. Micropillars provide
additional selection by bouncing off apoptotic bodies or larger
microvesicles [31] (Figure 3).

CLINICAL APPLICATIONS OF EXOSOMES
Exosomes as diagnostic tools
Exosomal protein and nucleic acid cargo strongly resemble
the intracytoplasmic components of the origin cell, offeringvaluable information in the form of biomolecules [6,32].
Exosomes can be isolated from any extracellular
fluid compartment, including blood, urine, pleural fluid,
bronchoalveolar lavage, cerebrospinal fluid etc. [1,3], providing
a promising, non-invasive alternative to biopsy. Exosomesreflect molecular changes that appear in the parental cells andmay function as biomarkers [1]. Moreover, extracellular vesicles’
number and cargo are cell-line specific and cell-status specific(physiological or pathological) [10].
Acute myeloid leukemia relapse originates from minimal
residual disease and correlates with a poor prognosis. Therefore,
early diagnosis and prevention of MRD is one of the main
objectives for improving the morbidity and providing longer
survival rates. Novel molecules with potential diagnostic role in
MRD include the exosomal miR-150, miR-155 and miR-1246.
MiR-1246 was also found in the tumor microenvironment as a
T-regs-recruiting molecule [6].
In digestive tract pathology, miR-19b-3p and miR-106a
isolated from serum exosomes were predictive for gastric cancer
and miR-7641 correlated with colorectal cancer evolution.
CD63+ exosomes isolated from urine of diabetic patientswas increased at the early stage of renal injury in diabetic
nephropathy [10].
Rats subjected to ischemia/reperfusion injury modelof acute kidney injury expressed a 60-fold increase of urine
exosomal levels containing miR-192 [8].
Exosomes with high EpCAM expression were found inpatients with early stage ovarian cancer compared to the control
group.

Fig. 3. Exosome isolation techniques. A. Ultracentrifugation involves
increasing the centrifugation speed up to 100,000-150,000 x g, when
exosomes can be extracted. The exosome suspension is not purebecause it is contaminated with nucleic acids and protein aggregates.
B. Gradient-density ultracentrifugation uses a density gradient ofsucrose/ iodixanol. Based on this density gradient, suspended particles
will migrate to their isopycnic point, allowing further separation. C.
Moving zone density gradient ultracentrifugation allows microvesicle
separation based on their size. D. Size-exclusion chromatography
principle. Particles that need to be isolated are suspended in a porous
stationary phase. Larger particles (blue) cannot pass between the
pores and they travel the fastest through the chromatography column.
Small particles (green) enter in the porous material and travel throughits microarchitecture, subsequently delaying their elution time. E.
Nanowire-based microfluidic separation system. Micropillars provide
support for the silicone nanowires and allow size exclusion for thelarger particles (blue). Exosomes (magenta) are trapped in the nanowire
network because the spacing between the wires is equal to the exosomal
diameter. Proteins or nucleic acids (green) pass through the nanowire
mesh easily and are rapidly eluted.
2021.31.1 (101 )  Fiziologia - Physiology

Finally, lung adenocarcinoma-specific exosomes foundin plasma were increased in patients with advanced disease
compared to healthy volunteers [1].
A new hope: exosomes as drug delivery vehicles
The biochemical composition of exosomes makes them asuitable candidate for drug delivery. The vesicular lipid bilayer
surrounds a hydrophilic core, an ideal medium for water-soluble
drug transport [7].
Some drugs that in theory have proven to have therapeutic
activity might not work in vivo due to their poor pharmacokinetics.
One good example is doxorubicin which is coated in liposomal
particles to improve its anti-tumoral efficacy and stability in theextracellular milieu. Exosomes, however, provide a better stability
in the extracellular space, a longer half-life (implying a larger
interval between administrations) and a lower immunogenicity [3].
Due to their small diameter (30-150 nm) [10], exosomes cancross the blood-brain barrier, making them a suitable candidatefor drug delivery inside the central nervous system [6,12]. Thomi9et al. demonstrated using Wistar rat pups that infrared labelled
MSC (mesenchymal stem cell) derived exosomes administeredintranasally prior to an ischemic challenge were uptaken in
the central nervous system starting from the frontal lobe (in30 minutes) and were distributed in the entire brain mass inapproximately 3 hours. MSC exosomes significantly reduced
neuronal and oligodendrocyte necrosis [33].
The exosomal surface protein repertoire provides additional
benefits including low immunogenicity (by FasL and galectin 9
expression [10]), escape from phagocytosis (due to high CD47
expression) and higher tissue- or organ-specificity [7].
Extracellular vesicles loaded with miRNAs or siRNAs
can suppress specific gene expression. Supporting evidence
proved that MSC-derived exosomes loaded with siRNA specificfor the KRAS gene, one of the most prominent genes involvedin pancreatic adenocarcinoma, reduced tumor size in a micemodel [3].
Zhu et al. found that NK-derived exosomes induce apoptosisof B16F10 melanoma cells in vitro and inhibited tumor growthin C57BL/6 mice [34].
Dendritic cell-derived exosomes express HLA class I andclass II molecules loaded with peptides facilitating immune
priming and effector T and NK cell activation [7]. Therefore,
DC-derived exosomes from convalescent patients can provide
a novel alternative in vaccine development [1,7,9]. In addition,phase I clinical studies in melanoma patients showed that
dendritic cell-derived exosomes have proven to be safe, nonallergic and associated with long-term stabilization and tumor
regression by increasing the circulating NK cells and NKG2D(NK group 2 member D)-dependent functions [1].
Drug loading techniques
Drugs can be loaded into exosomal particles either by
inducing changes in the parental cells or by modifying theextracellular vesicles after their isolation.
Pre-loading strategies involve drug delivery to the parental
cell and subsequently, the drug will be contained within the
exosomes at the moment of their release. Techniques include
viral transfection and liposomal drug delivery. One of the
major issues regarding these methods is that the drug or virus
can influence cell metabolism and signaling, leading to an
unexpected surface protein signature.
“Surface display” implies cellular transfection with avector that encodes a chimeric protein formed by an ubiquitous
exosomal membrane protein (LAMP2B, lactadherin etc.) fusedwith the desired peptide sequence that will be expressed on
the surface [3].
Post-loading methods imply that the drug is loaded after
the exosomes were isolated. In order to effectively load theexosomes, one must assure that the exosomal isolation was
done with a high yield and without biological function loss.
Techniques include electroporation, sonication and freezingthawing.
Electroporation is a technique in which an electric current
in applied to a cell suspension, resulting in cell membrane pore10

formation that allows internalization of drugs or nucleic acids
inside the cell. Wang et al. loaded A549 non-small cell lungcancer-derived exosomes with docetaxel using electroporation.
The drug delivering exosomes were administered to a micemodel of lung cancer and had a better outcome than the
docetaxel therapy alone [35].
Sonication relies on acoustic cavitation of the microbubbles
of exosomal suspensions exposed for a short time to ultrasounds.
Cavitation provides a transient membrane pore that allows drug
passage inside the cell [36].
CRISPR-cas9 loading
CRISPR-Cas9-loaded exosomes provide a promisingalternative to viral vectors. Main advantages include low
immunogenicity (the outer membrane is recognized as a selfentity) and high cell line specificity due to the similar surface
protein signature to the cell of origin [3].
Kim et al. obtained in a murine model of ovarian cancer asignificant decrease in tumor size in mice by loading sgRNA andcas9 plasmid into tumor-derived exosomes [37].

CONCLUSION
Initially considered only a cell disposal mechanism,
exosomal secretion has proven to be an effective, highly-specificcell communication strategy. This novel type of cell signaling was
found to be involved in physiological processes such as immune
priming, placental development or tissue regeneration, but alsoin pathologies such as cancer, diabetes or atherosclerosis.
Exosomal isolation is a growing research field for discovering
new separation techniques that preserve their physical properties
and biological functions.
Exosomes reflect their cell of origin as well as the cell’sfunctional status. Therefore, proteins or nucleic acids isolated
from extracellular vesicles can provide early diagnostic or
disease progression biomarkers.
Besides their diagnostic potential, drug-loaded exosomal
vesicles provide an organ-specific drug delivery, thereby reducing
potential adverse effects.
Due to their multivalent role in regulating, exosomes areindeed valuable players in health and disease.
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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ROLUL EXOZOMILOR ÎN PROCESELE FIZIOLOGICE ȘI
PATOLOGICE UMANE
REZUMATFuncționalitatea organismului uman presupune o interacțiune bine-coordonată între diferitele tipuri celulare. De-a lungul evoluției,organismele multicelulare și-au dezvoltat o varietate de căi de comunicare atât la nivelul micromediului local, precum și la distanță. Pe
lânga hormoni și interacțiunile dintre proteine, exozomii asigură o strategie de comunicare alternativă cu înaltă stabilitate și specificitate.
Penetrabiliatea crescută la nivelul organelor și țesuturilor se datorează imunogenicității și dimensiunilor reduse.
În acest studiu sunt abordate diferite aspecte legate de biogeneza exozomilor, rolul acestora în coordonarea diferitelor interacțiuni
fiziologice și patologice, strategii diverse de izolare a exozomilor, precum și potențialele lor aplicații în diagnosticul precoce și transportul
de medicamente.
Cuvinte cheie: exozom, inflamație, metastază, angiogeneză, diferențiere celulară, micromediu tumoral

12

Fiziologia - Physiology  2021.31.1 (101)PHYSICAL AGENTS AS ANTIMICROBIAL FACTORSCARMEN TATU1,2, GABRIELA TĂNASIE1,2, ALINA-GEORGIANA SIMINA2, CRISTINAGAȘPAR3, MARIUS GEORGESCU1, FLORINA BOJIN2,4, DANIELA PUȘCAȘIU5, CARMENPANAITESCU1,2
Department of Functional Sciences, Physiology, Center of Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babeș” University of Medicine
and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2, 300041 Timișoara
2
OncoGen Research Center, Timisoara County Emergency Clinical Hospital, Liviu Rebreanu Boulevard, no. 156, 300723, Timișoara
3
Banat’s University of Agricultural Science and Veterinary Medicine “King Michael the Ist of Romania” from Timisoara, Aradului str no. 119, 300645,
Timișoara, Romania
4
Department of Functional Sciences, Immunology, Center of Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babeș” University of
Medicine and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2, 300041 Timișoara
5
Department of Microscopic Morphology, Cellular and Molecular Biology, “Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania,
Eftimie Murgu Sq. no. 2, 300041 Timișoara
1

ABSTRACT
Antibiotic resistance to most antibiotics currently used is constantly on the rise, thus posing a serious threat to human health. Controlof microorganisms is essential to prevent the transmission of diseases and infections and to prevent unwanted microbial contamination.
Therefore, it is necessary to develop new strategies by which pathogenic bacteria could be eliminated. The spread of microorganisms cancontrolled by using physical and chemical agents. Physical agents include control methods such as high or low temperature, desiccation,
radiation, osmotic pressure, filtration. The result of the action of these factors is related to their intensity, the duration of time the bacterialcell was exposed, the environment in which it activates, the cell structure, but also its sensitivity to one or more factors. That is why it isnecessary to develop new strategies through which pathogenic bacteria can be easily annihilated, as alternatives to, or associated with,
antibiotic treatment.
Key words: physical agents, antimicrobial factors, temperature, desiccation, radiation, osmotic pressure, filtration.

INTRODUCTION
Antibiotic-resistant bacteria not only affect human health, butalso threaten the safety of patients in hospitals and communities.
However, the emergence of drug-resistant bacteria is inevitable
due to evolutionary selection but also as a consequence of thenon-discriminatory use of antibiotics. In pathogenic bacteria,
antibiotic resistance is constantly increasing compared to mostantibiotics currently used, thus posing a serious threat to humanhealth. Growing concerns about multiple drug resistance havealarmed the international scientific community. The World HealthOrganization (WHO) in its global report on antibiotic resistancepublished in 2014 stated that: “This serious threat is no longer a
prediction for the future, it is happening right now in every regionof the world and has the potential to affect anyone, of any age,in any country ”. The problem of antibiotic resistance is further
amplified by the horizontal transfer of resistance genes among
microbial populations. Consequently, not only is human healththreatened, but an environmental crisis can also be caused. For
example, the contamination of aquatic ecosystems and water
reservoirs with antibiotics causes the emergence of multidrug
resistance to an alarming level and directly results in increased
infection rates.
Therefore, it is necessary to develop new strategies bywhich pathogenic bacteria could be eliminated. Control ofmicroorganisms is essential to prevent the transmission of
diseases and infections and to prevent unwanted microbial

contamination. Microorganisms are controlled by physical andchemical agents. Physical agents include control methods,
such as high or low temperature, desiccation, radiation, osmoticpressure, filtration. Control by chemical agents refers to the useof disinfectants, antiseptics, antibiotics and chemotherapeutic
antimicrobial chemicals. Bacterial activity is influenced by a wide
range of physical, chemical and biological factors. The result of
the action of these factors is related to their intensity, the bacterialcell exposure time, the environment in which it activates, the cellstructure, but also its sensitivity to one or more factors.
Physical agents that can influence the development of
microorganisms are:
1. Temperature
Almost all microbial species have a temperature point
called “optimal” , at which they carry out their activity normally,
and certain limits below and above it, which when reachedhinder their development. Thus, from the point of view of the
optimal temperature, the bacteria are classified in the following
categories [1]:• mesophiles - pathogenic or conditioned pathogenic species
adapted to an optimal temperature of 37-38˚C, human bodytemperature, with limits between 25-34˚C / 10-45˚C;• psychrophiles (cryophiles) - which include microorganisms
whose optimal temperature is between 15-20˚C with limits
between 0 and 20˚C / 0-35˚C;• thermophilic - include garbage putrefaction bacteria whose
optimum temperature is between 50-53˚C with limits between
50-80˚C (85˚C - bacteria in geysers).
High temperature - going beyond the upper limit is harmful,

Received 17th of May 2021. Accepted 18th of June 2021. Address for correspondence: 2OncoGen Research Center, Timisoara County Emergency Clinical
Hospital, Liviu Rebreanu Boulevard, no. 156, 300723, Timișoara, Romania; e-mail: carmen.tatu@umft.ro2021.31.1 (101 )  Fiziologia - Physiology

13leading to disorganization of metabolic reactions throughprotein coagulation (the theory of protein denaturation andcoagulation) or by the accumulation of toxic products resulting
from biochemical processes (the intoxication theory). There aredifferences between the action mode of dry heat and wet heat[2]. In medical practice, heat is used in the process of sterilizing
medical instruments and materials used in laboratory diagnosis.
Sterilization is performed by dry heat (hot air, incineration) andwet heat (boiling, autoclaving and pasteurization).
a) It is harder for dry heat to penetrate the bacterial cellwall, unlike moist heat. Dry heat is less efficient - for
the Clostridium bacillus the temperature required todestroy is 140˚C at an exposure time between 5-15
mind, and a temperature of 180˚C for the Bacillus
anthracis at an exposure time of 60 minutes. Dry heatkills microorganisms through a process of proteinoxidation, rather than by coagulation of proteins.
Examples of dry heat sterilization include [1, 2]:• Hot air sterilization - very high temperature
dry heat is used: 171 ° C for 1 hour; 160 ° C
for 2 hours or more; or 121 ° C for 16 hours
or more, depending on volume. In general,they are only used to sterilize glassware,
metal tools and other inert materials, suchas oils and powders that are not damagedby the excessive temperature.
• Incineration - Incinerators are used todestroy disposable materials or consumables
by burning.
b) Wet heat penetrates more easily, due to thesoftening of the cell wall and the increase of its
permeability. Wet heat has a bactericidal effect onvegetative forms exposed at 100˚C for 2-3 ‘or at
60˚C for 1 hour. Bacterial spores are destroyed by
autoclaving for 15 ‘at 121˚C and at a pressure varying
between 1.5 atm - 2 atm. Autoclaving, therefore,
uses steam under pressure. Normally, water boils
at 100 ° C, but when put under pressure, the boilingtemperature rises to 121 ° C, enough to destroy thebacterial endospores. The same autoclaving process
is used to destroy hepatitis viruses that can survive
exposure to boiling water for up to 30 minutes, andthe endospores of certain species of Clostridium andBacillus can survive even hours of boiling [3].
c) Pasteurization is the gentle heating of milk andother materials to destroy certain microorganisms or
pathogens. However, it does not kill all microorganisms.
The milk is usually pasteurized by flash heating to 71.6
° C for at least 15 seconds, or 62.9 ° C for 30 minutesby the holding method.
Low temperature - generally inhibits microbial growth byslowing down the microbial metabolism, making bacteria kept
at low temperatures no longer multiply and enter latent life [4].
Exceptions to this rule are the meningococcus and gonococcus.
More intense cold, below minus 5-10˚C, preserves the biologicalproperties more efficiently than a temperature of 0˚C or - 10˚C.
Slow freezing damages bacteria to a greater extent than a sudden
freeze, because in the first case ice crystals form, which break thecellular structures and produce saline hyperconcentration with
distortion of the colloidal state of proteins. Repeated freezing
14

and thawing is harmful to microbes. Cold finds its applicationsin the preservation of food, some drugs and laboratory microbial
strains (reference bacterial cultures).
Numerous studies have aimed to track the effect of temperature
variations on microbial cultures. Thus, Cebrian et al. [5] studied
the influence of cultivation temperature on E. coli resistanceto 3 different agents: heat, pulsed electric field and hydrogen
peroxide. E. coli was grown in a stationary phase at 10 ° C, 20° C, 30 ° C, 37 ° C and 42 ° C. The obtained results showed an
influence of the cultivation temperature depending on the typeof stress applied. Cells subjected to a high culture temperature
were also resistant to heat, but were more sensitive to thepulsed electrical fields and hydrogen peroxide, and cells thatwere cultured at 10 ° C and 20 ° C were much more resistantto pulsed electrical field and hydrogen peroxide.
The results obtained during this investigation support the opinion
that the three agents studied act through different mechanisms,
due to the different inactivation kinetics observed. Despite
the differences between the action mode of the three agents
studied, they all involve the membrane at some point, which isan essential structure in bacterial survival under environmental
stress. Thus, it seems that cells with more fluid membranes, i.e.
those cultured at 10 ° C, would be more sensitive to the actionof heat, but more resistant to the formation of pores in the pulse
electrical field and the penetration of hydrogen peroxide intocytoplasm.
The study will have to continue in order to clarify the action
mechanisms of each agent, and in addition the role of othercellular structures that could be involved in the resistance andthe destruction of the cell must be considered. In conclusion,
the more heat-tolerant cells were not simultaneously resistantto oxidative stress and pulsed electrical field treatments.
2. Drying (desiccation)
Drying or desiccation in general has a static effect on
microorganisms because the lack of water inhibits the actionof microbial enzymes. Dehydrated and freeze-dried foods, forexample, do not require refrigeration because the absence ofwater inhibits microbial growth. The sensitivity of bacteria todehydration differs in relation to the species and even the strain.
Microorganisms such as meningococci, gonococci, treponemes,
leptospires are very sensitive. Tuberculosis bacilli, staphylococci
remain viable for long periods of time. Low temperature, protein
environment and the lack of oxygen considerably protect
microbes from the harmful effects of dehydration. Lyophilization(cryodesiccation) is based on these principles. The method
consists in freezing the microbial suspension in a protective
medium, drying it in vacuo and keeping it in closed ampoules
with inert gas. Vaccines and reference microbial strains arepreserved by lyophilization.
3. Radiation
Radiation acts on bacteria through various mechanisms:
the production of bactericidal effect peroxides in the environment(ionizing radiation: X, α, β, γ) and by direct effect on cellular
nucleic acids (solar radiation, ultraviolet).
Ionizing radiation (X, α, β, γ, neutrons) have strong bactericidal
action, being carriers of higher energies. They cause denaturationof proteins and cellular nucleic acids by inactivation of ribosomes,
rupture of nucleic acid chains, inactivation of enzymes due to
Fiziologia - Physiology  2021.31.1 (101)the formation of free radicals (OH, H) in the environment, theformation of peroxides (R-O-O-R) that act as oxidizing agents.
In small doses, they can produce bacterial mutations (X-rays)
with morphological and pathogenicity changes. They’re used forsterilization of medical instruments, medicine, food.
Ultraviolet radiation
The ultraviolet portion of the light spectrum includes allradiation with wavelengths from 100 nm to 400 nm. It has ashort wavelength and low energy. The bactericidal activity ofultraviolet (UV) light depends on the duration of exposure (thelonger the exposure, the longer the bactericidal activity) andthe wavelength of the UV rays used (range 260 nm - 270 nm isstrongly bactericidal because it acts by selective absorption at
the level of nucleic acids) [2].
The mechanism by which UV radiation destroys bacteria:
UV light is absorbed by microbial DNA and causes the adjacent
thymine bases on the same DNA strand to covalently bind toeach other, forming what is called thymine-thymine dimers. As
DNA replicates, the nucleotides are not complementary basepairs to the thymine dimers and this ends the replication of that
DNA strand.
Most of the damage caused by UV radiation comes fromthe fact that the cell tries to repair DNA damage through theprocess called “SOS repair”. In very strong DNA that contains
a large number of thymine dimers, the SOS repair process is
activated as a last effort to bring the DNA back to normal. A DNA
that contains many incorrectly embedded bases is synthesized.
In other words, UV radiation cause mutations and can lead topoor protein synthesis. With sufficient mutation, the bacterial
metabolism is blocked and the organism dies. Agents such as
UV radiation that cause high mutation rates are called mutagenic
agents. Radiation is used to sterilize rooms, boxes, plastics,
medicines.
Among the studies that used radiation to increase
antibacterial efficiency, is the one conducted by Chen et al in
2020. They used 2 strains of multi-resistant E. coli that wereisolated from urine samples collected in the clinical laboratory,
and tested these strains for antibiotic susceptibility. The firststrain, called MDR-1, was resistant to penicillin (ampicillin AMP), tetracycline (tetracycline - TETRA), quinoline (nalidixic
acid (nalixide)) and aminoglycosides (streptomycin - STR),
and the second isolated strain was also called MDR-2. it isresistant to penicillin (ampicillin - AMP), tetracycline (tetracycline- TETRA), quinoline (nalidixic acid (nalixide)) but also to otheraminoglycosides (spectinomycin, gentamicin), chloramphenicol.
Bacterial culture of E. coli MDR-2 left overnight was resuspended
in fresh LB broth with a final concentration of 10-7-10-8 CFU ml-1.
A volume of 10% nanocomposites (abbreviated graphene oxideGO, silver supported on abbreviated graphene oxide GO-Ag) or
water (in case of control) was added to the LB broth so that the
final concentration of the nanocomposites GO, GO-Ag is 3 and7μg ml-1, respectively. Before being subjected to laser beamirradiation (808 nm wavelength), the broth was stirred for one
hour at 37 ° C. During the irradiation period, the temperature
was measured with a thermal chamber, the irradiation time
being 7 minutes.
The treated mixed sample was cultured for 6 hours before
the bacterial viability was assessed. Among the many uses of
the GO-Ag nanocomposite, the authors reported its use forthe first time in combination with photo-thermal therapy. The
2021.31.1 (101 )  Fiziologia - Physiology

results showed an increase in antibacterial efficiency due tosilver nanoparticles that inhibit bacterial growth while graphene
oxide sheets absorb NIR light (near-infrared spectroscopy)
and generate heat. The conclusion of this study is a promising
one in terms of clinical utility. Compared to the widely used
AgNP, the GO-Ag nanocomposite showed a much better
antibacterial efficiency in clinically isolated multidrug-resistant
E. coli. Moreover, photothermal treatment could be combined
to lower the antibiotics dose and, in this way, multidrug-resistant
E. coli would be completely killed with reduced cytotoxicity.
Immunofluorescence images taken to visualize the results
showed that the integrity of the bacteria was compromised
after treatment with GO-Ag. Given the excellent antibacterial
performance of GO-Ag nanocomposites against widespread
multidrug-resistant E. coli bacteria, they could be used inconjunction with more common antibacterials in future medical
applications.
The effect of electromagnetic radiation with visible range
on E. coli samples was studied by Azeemi et al. (2017) in order
to find the right dose that could be used in the treatment ofdiseases caused by E. coli [6]. The strain was isolated from urine
samples obtained from hospitalized patients with Urinary Tract
Infection (UTI). The presence of E. coli in the obtained samples
was identified by standard laboratory methods, by performing
both morphological characterization and also biochemical tests.
A single colony of E. coli was inoculated into 3 ml of lysis broth.
From the suspension obtained, 40 μl were transferred to tubes
to be subjected to radiation of visible spectrum of different
wavelengths for 18 hours, after irradiation the samples were
kept in a stirrer incubator at a temperature of 37 ° C.
Statistical analysis was performed using an IBM SPSS,
version 20. A Pearson correlation coefficient was used toevaluate the relation between the optical densities of theirradiated samples and the number of colonies, obtaining astrong positive correlation between the 2 variables. The obtained
results showed a different response of the E.coli culture to the 6
waves, the deepest inhibitory effect was at 538 nm characteristicof the green color which proved to be bactericidal in contrast
to the wavelength of 453 nm characteristic of the blue color,the sample irradiated at this wavelength recording the largest
number of colonies.
Thus, it can be concluded that the radiation in the visible
region, i.e. 538 nm (green), 590 nm (yellow) can be effectively
used to treat diseases transmitted by E. coli. The effects ofvisible radiation on E. coli clearly show that different wavelengths
affected E. coli differently. All these results indicate that some
genetic changes lead to particular morphological behaviors.
The direct effect and determination of the optimal action
parameters of LED (blue-red and red-infrared radiation) on the
antibiotic sensitivity of the clinical isolates Staphylococcus aureus
and the tested strain Staphylococcus aureus ATCC 25923,
was studied by Pantyo and collaborators in 2019 [7]. These
researchers performed an experiment in which the radiation
exposure time of the studied cultures varied from 5 to 25 minuteswith an interval of 5 minutes and the radiation frequency was
0 Hz; 10 Hz; 600 Hz; 3000 Hz; 8000 Hz. The research results
showed an increase in the sensitivity of the microorganism tosome of the antibiotics tested. Based on the data obtained, theresearchers involved in the study have developed an algorithmand clinical recommendations for the use of this type of radiation15

in complex therapies that are involved in the treatment ofinflammatory diseases [7].
The conclusion of this study is that the degree of influenceof the applied radiation depends on the wavelength, duration andfrequency of radiation pulses - the most pronounced increase
in sensitivity was observed at exposure of 5 minutes at 0 Hz
(continuous radiation).
Abdulameer and Maki analyzed the effect of photodynamic
inactivation on the methicillin-resistant Staphylococcus aureus
strain using 2 different lasers: the so-called DPSSL (solid-statelaser pumping diodes) at a wavelength of 532 nm combined witha dye (Safranin O) and Laser Diode with a wavelength of 650
nm combined with methyl blue [8]. 100 samples from patientswith burns and infected wounds hospitalized in 2 hospitals in
Baghdad were tested for antimicrobial susceptibility using the
Kirby-Bauer method. The samples were divided into 4 categories:
a control category, a category focused on the photosensitization
reaction, a category subject to irradiation and a category in which
the photosensitization reaction was combined with the irradiationof the samples. The results obtained by the researchers
demonstrated the efficiency of the DPSSL laser, registering asmall number of cells. Regarding the exposure time, it took 3
minutes in the case of the DPSSL laser compared to 11 minutes
in the case of using the Diode Laser, the conclusion of theresearchers being that the use of photo-dynamic inactivation as
an alternative method in treating infected wounds and superficial
burns is viable.
Researchers led by Chaurasia A (2016) propose, following
the results obtained, a new treatment based on the action ofmagnetic nanoparticles against pathogenic bacteria, which block
the formation of biofilm and kill bacteria [9]. In this approach,the antibiotic-resistant Staphylococcus aureus strain (USA300)
and the uropathogenic Escherichia coli CFT073 are “trapped”
in the positively charged magnetic nanoparticles (MCSNP) byelectrostatic interaction leading to their complete destruction
within 30 minutes, due to the loss of membrane potential when
the sample is exposed to a radiofrequency current.
The obtained results demonstrate the efficiency of themethod, which can be used as an alternative antibacterial
strategy without leading to antibacterial resistance, andcan be used successfully in therapeutic applications. In thisexperiment, Escherichia coli strains (E. coli DH5α and E. coli
CFT073, UPEC) were cultured in Luria Bertani broth medium(LB) or LB-agar plates (1.5% agar) at 37 ° C. . Broth cultures
were grown under orbital shaking at 200 rpm, while the strainson the plates were grown in an incubator at 37 ° C overnight.
Methicillin-resistant Staphylococcus aureus (MRSA) strains
were cultured in tryptic soy broth (TSB) or brain-heart infusion
medium (BHI) under orbital shaking at 200 rpm or on tryptic
soy agar plates (TSA, 1.5% agar) at 37 ° C. Cell growth wasmonitored by measuring optical density at 600 nm (OD600).
Appropriate amounts of antibiotics such as ampicillin (Amp, 100
μg / mL), kanamycin (Km, 50 μg / mL), chloramphenicol (Cm,
33 μg / mL) or streptomycin (Sm, 50 μg / mL) were added to therecombinant E. coli cultures. For TSB cultures and TSA plates,
12.5 and 25 μg / ml chloramphenicol, respectively, were used to
culture the recombinant strain S. aureus USA300 FPR3757 thatstably expresses the luxBADCE (MRSA) genes integrated into
the genome. In order to capture the bacteria and subsequently
visualize the MCSNP complex bacteria using an external
16

magnetic field, researchers developed a paramagnetic ironoxide core coated with a silicon coating. The reasons for using
MCSNP (magnetic core-shell nanoparticles) are the following:• prevents the agglomeration of the NP iron core;• acts as a physical barrier to the nano-bio interface to preventthe biological reduction of Fe (III) to Fe (II) using NADPH, thus
reducing the generation of ROS by Fenton, Fenton-type reactions
and / or Haber-Weiss;• prevents passive internalization of NP by bacteria by increasing
the size (> 40nm);• provides a homogeneous interaction surface for the negatively
charged bacterial cell.
The group led by Chaurasia proposes a model to explain
the disturbances of the microbial membrane following theapplication of treatment with magnetic nanoparticles exposed
to radiofrequency (RMT) [9]. The changes that occur lead to thedeath of the bacterial cell. These results indicate that physicaltreatment based on MCSNP (magnetic core-shell nanoparticles)
can make for an alternative antibacterial strategy without risk ofantibiotic resistance, and can be used for several purposes, bothin therapeutic and environmental applications.
4. UltrasoundUltrasounds are vibration with a higher frequency thansound waves (over 16,000 Hz). They are obtained with the helpof piezoelectric crystals. Ultrasound has a strong bactericidal
effect, destroying the bacterial cell relatively fast. In fact,
one of the most effective methods of disintegrating microbial
bodies - the preliminary stage of chemical analysis of bacteria
in the laboratory - is the use of ultrasound on the bacterial
suspension. The ultrasonic wave, when passing through extra
and intracellular water, determines intense movement of the
protoplast with the disintegration of cellular structures (breakingof the cell wall). It is used for disinfection and cleaning of dental
and surgical instruments, for chemical analyzes determining theseparation of cellular enzymes.
A 2019 study in the International Journal of Hyperthermia
aimed to investigate the bactericidal effects of high-intensity
focused ultrasound (HIFU) on Bacillus Calmette-Guerin (BCG, a
substitute for Mycobacterium tuberculosis) [10]. The conclusion
was that HIFU has the therapeutic potential to treat BCG-infected
tissues in rats and that a combination of mechanical, cavitationand thermal effects most effectively inactivate BCG bacteria
through HIFU. The hope of this study was to provide a plausible
basis for an effective non-invasive treatment for tuberculosis.
5. Osmotic pressure
Microorganisms, in their natural environment, are constantly
facing changes in osmotic pressure. Water tends to flow throughsemipermeable membranes, such as the cytoplasmic membraneof microorganisms, to the part with a higher concentration ofdissolved materials. In other words, water goes from a higher
concentration of water to a lower concentration of water. If theconcentration of dissolved substances is higher inside the cellthan outside, the cell is in a hypotonic environment, which attracts
water into the cell, but the rigid cell walls of bacteria and fungi,
however, prevent their explosion or plasmoptysis.
When the concentrations of dissolved substances are thesame both inside and outside the cell, the cell is said to be in
an isotonic environment. Water passes evenly into and out of
Fiziologia - Physiology  2021.31.1 (101)the cell. Hypotonic and isotonic environments are not usuallyharmful to microorganisms. However, if the concentration ofdissolved materials is higher outside the cell than inside, then thecell is in a hypertonic environment. Under this condition, water
leaves the cell, resulting in cytoplasmic membrane contraction
or plasmolysis. Under such conditions, the cell dehydrates andits growth is inhibited. Young vegetative cells are more sensitive
than spores [11].
6. Electricity
Electricity has minor effects on bacteria, but can causechanges in the environment (physico-chemical) with bactericidal
side effects: rising temperature, release of chlorine fromcompounds, ozone formation.
7. Filtration
Filtration is performed by passing a fluid through a porous
body (filter). Depending on the pore diameter, the filtration can beclarifying or sterilizing. Microbiological membrane filters provide
a useful way to sterilize materials such as vaccines, antibiotic
solutions, animal sera, enzyme solutions, vitamin solutions
and other solutions that may be damaged or distorted by hightemperatures or chemicals [12].
Filters contain pores small enough to prevent the passageof microbes, but large enough to allow fluid to pass through the
body. The liquid is then collected in a sterile flask.
Filters with pore diameters from 25 nm to 0.45 μm areusually used in this procedure. A membrane with a porosity of
0.22μm retains all bacteria. Filters can also be used to remove
microorganisms from water and air for microbiological testing.

Influence of led radiation on the Staphylococcus aureus
sensitivity to antibiotics, Photobiol Photomed, 2019, 26,
50–55. 10.26565/2076-0612-2019-26-07
8. Abdulameer FH, Maki MA, Antibacterial Photodynamic
Effect of 532 nm Diode-Pumped Solid State and 650 nm
Diode Lasers on Methicillin Resistant Staphylococcus
Aureus in Vitro, Iraqi Journal of Laser, 2017, Part B, Vol.
16, pp.21-26
9. Chaurasia A, Thorat N, Tandon A, Kim JH, Park HS., Kim
KK, Coupling of radiofrequency with magnetic nanoparticles
treatment as an alternative physical antibacterial strategy
against multiple drug resistant bacteria, Sci Rep, 2016, 6,
33662 . https://doi.org/10.1038/srep33662
10. Shuang X, Hua C, Jianhu L, Vishnu Prasad A, Min Y, Yu D,
Dairong L, Yonghong D, Bactericidal effects of high intensity
focused ultrasound on Bacillus Calmette-Guerin in vivo
and in vitro, Int J Hyperthermia, 2019; 36(1):886-896. doi:
10.1080/02656736.2019.1649474.
11. Kiamco MM, Atci E, Mohamed A, Call DR, Beyenal H,
Hyperosmotic Agents and Antibiotics Affect Dissolved
Oxygen and pH Concentration Gradients in Staphylococcus
aureus Biofilms, Appl Environ Microbiol, 2017 Mar
2;83(6):e02783-16. doi: 10.1128/AEM.02783-16.
12. Vonberg RP, Rotermund-Rauchenberger D, Gastmeier
P, Reusable terminal tap water filters for nosocomial
legionellosis prevention, Ann Hematol, 2005; 84(6):403-5.
doi: 10.1007/s00277-004-1000-6.

CONCLUSION

In conclusion, it is necessary to develop new strategies asalternatives to or associated with antibiotic treatment, throughwhich pathogenic bacteria can be easily annihilated.

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2.

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4.

5.

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Paluch E, Rewak-Soroczyńska J, Jedrusik I, Mazurkiewicz
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Cebrian G, Sagarzazu N, Pagan R, Condon S, Manas P,
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to various enviromental stresses. Journal of Applied
Microbiology, 2008, doi:10.1111/j.1365-2672.2008.03745.x
Azeemi YTS, Shaukat FS, Azeemi SK, Khan I, Mahmood K,
Naz F, Effect of visible range electromagnetic radiation on
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Pantyo VV, Pantyo VI, Gulyar SA, Koval GM, Danko EM,

2021.31.1 (101 )  Fiziologia - Physiology

17

AGENȚII FIZICI CA FACTORI ANTIMICROBIENI
RezumatRezistența la antibiotice este în continuă creștere față de majoritatea antibioticelor utilizate în prezent, astfel, constituind o amenințare
gravă pentru sănătatea umană. Controlul microorganismelor este esențial pentru a preveni transmiterea bolilor și infecțiilor și pentru a
preveni contaminarea microbiană nedorită. Prin urmare, este necesară dezvoltarea de noi strategii prin care bacteriile patogene ar putea
fi eliminate. Răspândirea microorganismelor poate fi controlată prin intermediul agenților fizici și al agenților chimici. Agenții fizici includ
metode de control, cum ar fi temperatura ridicată sau scăzută, desicarea, radiațiile, presiunea osmotică, filtrarea. Rezultatul acțiunii acestor
factori este legat de intensitatea acestora, de timpul de expunere a celulei bacteriene, mediul în care activează, structura celulei dar și
sensibilitatea ei la unul sau mai mulți factori. De aceea se impune dezvoltarea de noi strategii ca alternative ale tratamentului antibiotic
sau asociate cu acesta, prin care bacteriile patogene sa poata fi anihilate cu usurinta.
Cuvine cheie: agenții fizici, factori antimicrobieni, temperatura, desicarea, radiațiile, presiunea osmotică, filtrarea.

18

Fiziologia - Physiology  2021.31.1 (101)ASSESSMENT OF HEART RATE CHANGES TO POSTUREAND ORTHOSTATIC TOLERANCE IN A COHORT OF MALE
NIGERIAN UNDERGRADUATES
OLUSOJI A. ADALUMO1*, SAHEED A. LAWAL1, FUNMILAYO M. OWOLABI2
12

Department of Physiology, School of Health and Health Technology, Federal University of Technology, Akure (FUTA), Nigeria
Department of Biomedical Technology, School of Health and Health Technology, Federal University of Technology, Akure (FUTA), Nigeria

ABSTRACT
Background: Orthostatic tolerance and Postural Orthostatic Tachycardia Syndrome (POTS) has been shown to be a common disorderamong young adults. We suspect that the prevalence of POTS and orthostatic intolerance among Nigeria young population is likely
underreported owing to underdiagnosis. This study assessed orthostatic tolerance and the prevalence of postural orthostatic tachycardia
syndrome (POTS) in a cohort of male young adult population of a University.
Methods: Thirty undergraduate male students of the Federal University of Technology Akure (FUTA) aged 15 to 35 years (mean, 21.83± 2.82 years) were recruited for the study. The height and weight were measured using standard procedures. The blood pressure andheart rate were evaluated during supine and standing position using a digital sphygmomanometer. The postural heart rate difference(PHRD) was evaluated using established protocol.
Results: The mean ± SD of age, height, weight, BMI, BSA of the participants were 21.83 ± 2.82 years, 1.71 ± 0.06 m, 61.72 ± 7.22
kg, 21.1 ± 1.83 kg/m2, 1.79 ± 0.12 m2 respectively. The Supine Heart Rate (HRsup), Standing Heart Rate (HRsta), and Postural Heart
Rate Difference (PHRD) were 65.27 ± 9.44, 83.90 ± 13.09, and 18.63 ± 9.64 bpm respectively. Further, the Pulse rate (PR), Systolic
Blood Pressure (SBP), Diastolic Blood Pressure (DBP), Mean Arterial Pressure (MAP), Pulse Pressure (PP) and Rate Pulse Pressure(RPP) of the participants were 70.77 ± 10.25 bpm; 115.2 ± 9.62; 74.40 ± 9.09; 88.00 ± 8.17; 40.8 ± 9.27 mmHg respectively. Orthostatic
intolerance and POTS were observed in 13% of the participants.
Conclusion: These data suggest that orthostatic intolerance with POTS could emerge as a prevalent disorder with health consequences
within the Nigerian young population.
Keywords: Tachycardia, Posture, Pressure, Orthostatic Tolerance, Sympathetic, Autonomic, Physiology, POTS

INTRODUCTION

Postural orthostatic tachycardia syndrome (POTS) is anautonomic dysregulation categorized as a disproportionate
tachycardia upon standing in the manifestation of orthostatic
intolerance [1, 2, 3]. Present adult diagnostic criterion involves
a heart rate rise of greater than or equal to 30 bpm within
the early 10 minutes of standing or head-up tilt (HUT) in theabsence of orthostatic hypotension [4, 5]. Postural orthostatic
tachycardia syndrome primarily affects premenopausal females
between 15 and 50 years of age, expressing with symptoms
of fatigue, headache, sleep disturbance, palpitations, nausea,
bloating, and so on [2, 6, 3].
Several pathophysiologic mechanisms; including but notlimited to disproportionate sympatho-excitation, autoimmune
dysfunction, volume depletion, cardiac and physicaldeconditioning point to a heterogeneously complex etiology[3, 5, 7]. Most considerably, the devastating nature of postural
orthostatic tachycardia syndrome predisposes individuals to ahigh degree of physiologic dysfunctions and reduced value oflife, necessitating more population studies into this classicalphysiological event of clinical importance [8].
The underlying pathophysiology of postural orthostatic
tachycardia syndrome is heterogeneous, encompassing
excess sympathetic tone, impaired peripheral autonomic
function, cardiovascular deconditioning, volume dysregulation,
and autoimmune dysfunction [9]. In apparently healthy
subjects, a shift of the intravascular capacity to the interstitialspace decreases the overall effective circulating blood volume,
showing the gravity-dependent physiology seen in orthostaticrelated pathologies.
As expected, a succeeding decrease in stroke volume
results in a compensatory increased sympathetic drive
augmenting cardiac contractility, heart rate, and systemic
vascular resistance. Postural orthostatic tachycardia syndrome
patients display persistent reduced stroke volume despite an
extravagant sympathetic response with postural changes suchas standing, resulting in a final common pathway of tachycardia
in the existence of orthostatic intolerance on standing [4, 7].

Received 30th of April 2021. Accepted 29th of June 2021. Address for correspondence: Olusoji A. Adalumo, Department of Physiology, School of Healthand Health Technology, Federal University of Technology, Akure (FUTA), Nigeria; phone/fax: +2347031083977; e-mail: oaadalumo@futa.edu.ng

2021.31.1 (101 )  Fiziologia - Physiology

19

METHODS

ParticipantsThis was a cross-sectional descriptive study. The target
population was apparently healthy undergraduate students
within the age limit of 15 years and 35 years in The Federal
University of Technology, Akure. Inclusion criteria were; male,apparently healthy, undergraduate student, Body mass index <30. Exclusion criteria were; female, suffering from any chronic
cardiac disease, less than 15 years of age, greater than 35
years of age, hypertensive, smoker, chronic alcohol taker. A totalof 30 volunteers (only males) selected by purposive samplingtechnique participated in the study.
Anthropometric Measurements
The height (m) and weight (kg) were measured using a
stadiometer (Liam Medical England, Model: NO. RGZ-160)
coupled with a weighing scale. The body mass index (BMI)
and body surface area (BSA) was determined as described
elsewhere [17, 18] calculated as follows:
BMI = weight (kg) / [height (m)] 2 [10].
BSA = [11].
Blood Pressure and its derivatives
The systolic blood pressure (mmHg), diastolic blood pressure(mmHg) and pulse rate (beat per minute) were measured using
the digital sphygmomanometer (Omron M2 Eco Automatic Upper
Arm Blood Pressure Monitor). The inflatable cuff was tied aroundthe left arm of the subjects and the sphygmomanometer recorded
the systolic blood pressure, diastolic blood pressure and pulse
rate. The systolic blood pressure, diastolic blood pressure andpulse rate were determined at supine position, sitting positionand standing position. The mean arterial pressure (MAP), pulsepressure (PP) and rate pressure product (RPP) were determined
as follows:
MAP = [(2 x DBP) + SBP] / 3 [12]Where, MAP is Mean Arterial Pressure in mmHg, DBP is Diastolic
Blood Pressure in mmHg and SBP is Systolic Blood Pressurein mmHg.
PP = SBP – DBP [13]
Where, PP is pulse pressure in mmHg, DBP is diastolic bloodpressure in mmHg and SBP is systolic blood pressure in mmHg.
RPP = HR X SBP [14]Where, RPP is Rate Pressure Product in mmHg per minute, HR
is the heart rate in beat per minute and SBP is the systolic bloodpressure in beat per minute.
Heart rate
The heart rate of the participants were determined at supine
and standing position (within 3 to 5 minutes of standing), using
a digital sphygmomanometer.
Postural Heart Rate Difference
Postural Heart Rate Difference (PHRD) is determined as theStanding Heart Rate minus the Supine Heart Rate.
PHRD = HRsta - HRsup
Statistical Analysis
All data collected were entered into Microsoft Excel 2013
and imported into SPSS’17 statistical software for analysis. Data
were processed using descriptive and inferential analysis. The
20level of confidence was at p < 0.05.
RESULTS
Anthropometric and Socio-demographic Characteristics
The study group was made up of 30 students (all males). The
mean age was 21.83 ± 2.82, with the minimum age of 16 andmaximum age of 30. The mean height was 1.709 ± 0.06 meters,with the minimum height of 1.56 meters and maximum heightof 1.87 meters. The mean weight was 61.72 ± 7.22 kg, withthe minimum weight of 49 kg and maximum weight of 80.5.
The mean Body Mass Index (BMI) was 21.1 ± 1.83, with theminimum BMI of 17.83 and maximum BMI of 25.99. The mean
Body Surface Area (BSA) was 1.79 ± 0.12, with the minimum
BSA of 1.46 and maximum of 1.98. All the participants werestudents of The Federal University of Technology, Akure. The
descriptive data is presented in table 1 below.
Heart Rate Response to Posture
The mean heart rate during supine and standing positions
were: 65.267 ± 9.436 and 83.90 ± 13.092 respectively. The
mean postural heart rate differences (PHRD) was 18.63 ± 9.64.
Orthostatic intolerance and Postural Orthostatic Tachycardia
Syndrome (POTS) was observed in 13.3% of the subjects. The
criteria for POTS was increase in heart rate with greater than orequal to 30 beat per minute within 3 minutes of standing fromsupine position. The descriptive data of heart rate response toposture is presented in table 3 below while the prevalence of
postural orthostatic tachycardia syndrome is represented infigure1.
Table I: Descriptive Statistics of Anthropometric and SocioDemographic Characteristics
S/nVariables

Mean ± SD

Minimum

Maximum1.

Age (Years)

21.83 ± 2.82

16

30

2.

Height (m)1.71 ± 0.06

1.56

1.87

3.

Weight (kg)61.72 ± 7.22

49

80.5

4.

BMI (kg/m2)

21.1 ± 1.83

17.83

25.99

5.

BSA (m2)

1.79 ± 0.12

1.46

1.98

Fiziologia - Physiology  2021.31.1 (101)Table II: Descriptive Statistics of Blood Pressure Parametersand Its Derivatives
S/nVariables

Mean ± SD

Minimum

Maximum

1.

SBP (mmHg)

115.2 ± 9.62

98

133

2.

DBP (mmHg)

74.40 ± 9.09

50

88

3.

PR (bpm)

70.77 ± 10.25

52

92

4.

MAP (mmHg)

88.00 ± 8.17

67.33

102.33

5.

PP (mmHg)

40.8 ± 9.27

26

60

6.

RPP (mmHg)

8173.17 ± 1519.70 5400

12144

Table III: Descriptive Statistics of Heart Rate Response to
PostureMinimum

MaximumSupine Heart Rate(bpm)
65.27 ± 9.44

48

92

2.

Standing Heart Rate(bpm)

83.90 ± 13.09

63

112

3.

PHRD (bpm)

18.63 ± 9.64

1

47

S/n

Variables

1.

Mean ± SD

DISCUSSION

The result shows 13.30 % prevalence of postural orthostatic
tachycardia syndrome (POTS) among undergraduate students
at The Federal University of Technology, Akure. POTS isa disorder in which the autonomic nervous system fails tocompensate for the upright position of the body upon standing
from supine position [15]. When lying in supine position, up
to 30% of the blood volume is occupied in the thorax. During
standing, 300–800 mL of blood is pulled downwards from thethorax into the abdomen and lower extremities due to force ofgravity. Most of this pooling into lower extremities veins occurs
within 10 seconds of standing. This results in a decrease invenous return to the right side of the heart with a subsequent
decrease in the stroke volume and cardiac output. Reductions
in pulse pressure and stroke volume are detected by arterialbaroreceptors (located in carotid sinuses and the aortic arch)and cardiopulmonary mechanoreceptors (located in heart andlung). Compensatory reﬂexes results in elevated sympathetic
nervous system output (peripheral arteriolar vasoconstriction)and depressed parasympathetic nervous system output (reduced
vagal tone to the heart with cardio-acceleration) [16]. After
standing in normal subjects, there is a 10–15 bpm increase inheart rate, but in people with POTS, the increase in heart rateis exaggerated to about 30 beats per minutes or more. Elevatedheart rate in POTS patients is due to exaggerated sympathetic
nervous system which can be attributed to any of the following:
autonomic dysfunctions, hyperadrenergic response to standing,
genetic composition of individuals, hypovolemia, and impaired
cerebral autoregulation [16]CONCLUSION

These data suggest that orthostatic intolerance with POTS
could emerge as a prevalent disorder with health consequences
within the Nigerian young population. Hence more studies
are warranted to determine the prevalence within the larger
population and geographical locations.

ETHICAL CONSIDERATIONS

Ethical standards regarding studies on human subject werestrictly followed. Informed consent of each participant before andthroughout the study were obtained in accordance with standardpractice concerning research relating to human subjects.

CONFLICTS OF INTEREST

Authors declare that there are no conflicts of interest concerning this publication whatsoever.

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Fig. 1: Prevalence of postural orthostatic tachycardia syndrome(POTS).
2021.31.1 (101 )  Fiziologia - Physiology

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J Health Sci 2015; 15: 71-4.
18. Ogunlade O, Adalumo OA. Mean Values, Normal Limits andSex Differences of Anthropometry of Young Adults in a University
Community in Nigeria. Amer J Cli Exp Med; Vol. 3, No. 1, 2015, pp.
44-47. doi: 10.11648/j.ajcem.20150301.16

22

Fiziologia - Physiology  2021.31.1 (101)EVALUAREA MODIFICĂRILOR RITMULUI CARDIAC LA
POSTURA ȘI TOLERANȚA ORTOSTATICĂ LA O COHORTĂ
DE STUDENȚI NIGERIENI DE SEX MASCULINREZUMAT
Toleranța ortostatică și sindromul tahicardiei ortostatice posturale (POTS) reprezintă o tulburare frecventă la adulții tineri. Se presupune căprevalența POTS și a intoleranței ortostatice în rândul populației tinere din Nigeria este probabil subdeclarată din cauza subdiagnosticului.
Acest studiu a evaluat toleranța ortostatică și prevalența sindromului de tahicardie ortostatică posturală (POTS) într-o cohortă de populație
adultă tânără de sex masculin dintr-o universitate.
Metode: 30 de studenți de la Universitatea Federală de Tehnologie Akure (FUTA) cu vârste cuprinse între 15 și 35 de ani (medie, 21,83± 2,82 ani) au fost recrutați pentru studiu. Înălțimea și greutatea au fost măsurate utilizând proceduri standard. Tensiunea arterială și
frecvența cardiacă au fost evaluate în decubit dorsal și în ortostatism, utilizând un sfigmomanometru digital. Diferența de ritm cardiac
postural (PHRD) a fost evaluată utilizând protocolul stabilit.
Rezultate: Media ± SD a vârstei, înălțimii, greutății, IMC, BSA a participanților a fost de 21,83 ± 2,82 ani, 1,71 ± 0,06 m, 61,72 ± 7,22kg, 21,1 ± 1,83 kg / m2, 1,79 ± 0,12 m2, respectiv. Ritmul cardiac în decubit (HRsup), ritmul cardiac în picioare (HRsta) și diferența defrecvență cardiacă posturală (PHRD) au fost de 65,27 ± 9,44, 83,90 ± 13,09 și respectiv 18,63 ± 9,64 bpm. Mai mult, rata pulsului (PR),tensiunea arterială sistolică (SBP), tensiunea arterială diastolică (DBP), presiunea arterială medie (MAP), presiunea pulsului (PP) și
presiunea pulsului (RPP) a participanților au fost de 70,77 ± 10,25 bpm; 115,2 ± 9,62; 74,40 ± 9,09; 88,00 ± 8,17; 40,8 ± 9,27 mmHg,
respectiv. Intoleranța ortostatică și POTS au fost observate la 13% dintre participanți.
Concluzie: Aceste date sugerează că intoleranța ortostatică cu POTS ar putea apărea ca o tulburare prevalentă cu consecințe asuprasănătății în cadrul populației tinere nigeriene.
Cuvinte cheie: Tahicardie, Postură, Presiune, Toleranță ortostatică, Simpatică, Autonomă, Fiziologie, POTS

2021.31.1 (101 )  Fiziologia - Physiology

23

EXPLORATION OF THE ASSOCIATION BETWEEN CLASSICALAND EMERGING RISK FACTORS FOR DIABETIC KIDNEYDISEASE AND ALBUMINURIA IN A COHORT OF TYPE 2
DIABETES MELLITUS PATIENTSMARCEL STOIȚĂ1,*, COSMIN MIHAI VESA1,2, AMORIN POPA1,2
1
2

Faculty of Medicine and Pharmacy, University of Oradea, Romania
Clinical County Emergency Hospital of Oradea, Romania
ABSTRACTThe presence of albuminuria in patients with type 2 diabetes mellitus is a marker of endothelial dysfunction and also one of the criteria
for diagnosing diabetic kidney disease. The present study aimed to identify associations between cardiovascular risk factors and renal
albumin excretion in a group of 218 patients with type 2 diabetes mellitus. HbA1c values, systolic blood pressure, diastolic blood pressure
were statistically significantly higher in patients with microalbuinuria or macroalbuminuria compared to patients with normoalbuminuria(p <0.01). We identified a statistically significant positive association between uric acid values and albuminuria, respectively 25- (OH)2
vitamin D3 deficiency and microalbuminuria (p <0.01).
Keywords: diabetes, microalbuminuria, hyperuricemia, vitamin D.

1. INTRODUCTION

Patients with diabetes mellitus are about 2 times more likely
to suffer from chronic kidney disease than people who do nothave diabetes (1). In the NHANES study in patients evaluatedfrom 2009 to 2014, the prevalence of chronic kidney diseasein patients with diabetes mellitus was 26.2%, the diagnostic
criteria being the presence of albuminuria (2). In diabetes
mellitus patients over 65 years of age, the prevalence of reduced
glomerular filtration rate was higher than in young patients, while
albuminuria had comparable prevalence in both categories. The
prevalence of chronic kidney disease in patients with diabetes
mellitus in 2008 in Shanghai, China was 33.5% (3). In Singapore
the prevalence of chronic kidney disease in patients with diabetes
was 53%, in 21% of patients a glomerular filtration rate <60mL/
min/1.73m2 was identified, 48% had albuminuria ≥30mg/g, and
28% they had diabetic retinopathy (4). In literature the factors thatwere associated with diabetic kidney disease were the presenceof diabetic neuropathy, hypertension, hypertriglyceridemia,
increased BMI, increased duration of diabetes, HbA1c≥8% andthe presence of cardiovascular disease.
Hyperuricemia is being more frequently recognized as arisk factor for diabetic kidney disease or (5). Numerous studies
indicate that hyperuricemia is independently associated withchronic kidney disease (6,7,8,9). According to a cross-sectional
study that included 2,108 Chinese patients with type 2 diabetes,

there was a positive correlation between uric acid values andalbuminuria and impaired renal function (10). Another study
demonstrated that during a 5-year follow-up of 1,449 type 2
diabetic patients with normal renal function, the presence ofhyperuricemia was identified as an independent risk factor forthe development of diabetic kidney disease (11). Although thepathogenesis of diabetic kidney disease has not been fully
investigated, it is currently thought to be related to glucose andlipid metabolism disorders, renal hemodynamic abnormalities,oxidative stress, elevated vasoactive substances, cytokinesand genetic factors. Numerous studies have identified a clear
correlation between vitamin D deficiency and type 2 diabetes,and vitamin D deficiency is quite common in patients with chronic
renal failure (12). Therefore, numerous risk factors for diabetic
kidney disease are being recognized contributing to a more
profound understanding of this disease.
The present study aims to evaluate the correlation
between albuminuria in patients with type 2 diabetes and uricacid and the correlation between albuminuria and vitamin D.

2. MATERIAL AND METHOD

The present study included patients with type 2 diabetes
who were evaluated at the County Clinical Hospital in Oradea,
Bihor. The inclusion criteria applied for this investigation were:age between 18 and 75 years, type 2 diabetes confirmed by
HbA1c determination, individuals who agreed to be investigated.
The exclusion criteria were: patients with glomerular filtration
rate <15 mL/min/1.73m2 or those on dialysis, patients with

Received 21st of February 2021. Accepted 16th of April 2021. Address for correspondence: Marcel Stoiță, Faculty of Medicine and Pharmacy, Universityof Oradea, Romania, Romania; e-mail: stoitamarcel2020@gmail.com

24

Fiziologia - Physiology  2021.31.1 (101)liver cirrhosis, patients with neoplastic diseases, patients withmental illness, patients with hypertension grade III defined as
SBP≥180mmHg or DBP≥120mmHg, patients suffering fromglomerular or tubular diseases, patients with HbA1c >12%,
patients with acute or chronic urinary tract infections. The study
was done between September 1, 2019 and March 31, 2020. A
number of 299 were initially considered eligible for inclusion in
the study but after applying the inclusion and exclusion criteria
only 218 of them remained in the study. The research was
conducted with the approval of the Ethics Commission of Oradea
County Hospital. The glomerular filtration rate was calculatedusing the CKD-EPI equation. In addition to the usual laboratory
determinations like HbA1c, lipid profile, creatinine, urea, it wasdone the determination of the albumin/creatinine ratio in theurine spot, the serum value of uric acid and the serum value of
1,25-(OH)2-vitamin D3. Turbidimetry was used for determinationof albumin/creatinine ratio, determination of 1,25-(OH)2-vitamin
D3 was performed by chemiluminescence and uric acid bycolorimetric enzymatic method. All the examinations mentioned
above were performed in the hospital laboratory. The presenceof microalbuminuria or macroalbuminuria was considered adiagnostic criterion for impaired renal function, we did not use
the diagnosis of chronic kidney disease because we could not
document the presence of a persistent elevated albuminuria.
Microalbuminuria was defined as estimated albumin/creatinine
ratio on between 30-300mg/g and macroalbuminuria as albumin/creatinine ratio ≥300mg /g. Values of​​ 1,25-(OH)2-vitamin D3 >50
nmol/L were considered normal and vitamin D deficiency wasdefined as 1,25-(OH)2-vitamin D3 ≤50 nmol/L. Hyperuricemia
was defined as the serum value of uric acid ≥7mg/dL. Good
control of glycaemic values was​​ defined as HbA1c <7%. A binary
variable was defined, the deficiency of 25-(OH)2-vitamin D3,
with a value of 1 if 1,25-(OH)2-vitamin D3 ≤50 nmol/L and 0 if
1,25-(OH)2-vitamin D3 >50 nmol/L.
Statistical processing was performed using Biostat software. The
average values o​​ f the different parameters were compared using
the Anova test. A value of p <0.05 was considered statisticallysignificant. Univariate regression and multivariate regression
were used to assess correlation between variables.

Urea (mg/dL) 18.38 ± 4.38 17.53±4.08

19.43±5.19

0.15

HbA1c (%)

7.80 ± 1.38

8.35±1.49

8.32±1.28

0.0088Albumin/creatinine ratio(mg/g)

19.99±(15.75,24.85)

80.71 (39.93,90.84)

5 6 9 . 4 3(354.57,640.78)

0.0001

BMI (kg/m2)

26.78 ± 3.91

27.34±3.89

28.95±4.19

0.31Hypertension
(%)

45.16

59.61

90.00

0.0001

SBP (mmHg)

128.5514.03

135.25±16.49

141.35±15.56

0.0026

DBP (mmHg)

79.23 ± 9.25

83±10.51

87.05±9.01

0.0087

GFR (mL/
min/1.73m2)

105.11 ± 11.48

105.83±10.63

99.01±16.88

0.67

1,25-(OH)2
Vitamin D3(nmol/L)

45.40 ± 16.16

40.54±13.84

38.92±18.13

0.045

Uric acid (mg/dL)

5.49 ± 1.22

5.55±1.54

6.27±1.53

0.76

Diabetes duration

6.12 ± 4.94

6.38±5.79

8.03±6.17

0.74

Colesterol (mg/dL)

180.8540.73±

186.89±41.21

173.24±41.76

0.68

LDL-cholesterol (mg/dL)

123.4031.63±

121.59±37.18

103.8±26.33

0.77

Tryglicerides(mg/dL)175.20 ±(94.76,183.33)

2 0 1 . 6 4(99.19,232.27)

2 7 5 . 9 8(123.99,266.81)

0.26

HDL-cholesterol (mg/dL)

42.59 ± 11.05

43.18±12.44

39.38±12.51

0.72

±

Correlation between uric acid values and albumin/creatinineratio
In figure 1 it is shown that there is a statistically significant
correlation between the value of serum uric acid and the urinary
albumin/creatinine ratio. High uric acid levels are associated withelevated urinary albumin/creatinine ratio (p=0,01).

3. RESULTS AND DISCUSSION
Table 1. Clinical-biochemical characteristics according to thelevel of albuminuria
NormoalMacroalbumiMicroalbuminuria
buminuria
nuria(n=104)(n=94)(n=20)

pAge

48.12 ± 8.71

50.78±8.5650.25±8.36

0.03

sex (%men)

67.74%

50.96%

65.00%

0.01

Creatinine(mg/dL)0.74 ± 0.16

0.68±0.16

0.80±0.22

0.098

Parameter

2021.31.1 (101 )  Fiziologia - Physiology

Fig.1. Correlation between serum uric acid value and albumin/creatinine ratio
25Correlation between 1,25-(OH)2-vitamin D3 values andalbumin/creatinine ratio
In figure 2 it is shown that there is no statisticallysignificant correlation between serum 1,25-(OH)2-vitamin D3
and urinary albumin / creatinine ratio (p=0.13).

Fig. 2. Correlation between serum 1,25-(OH)2-vitamin D3 value
and albumin/creatinine ratio

Table 2. Linear regression parameters between serum uricacid value and albumin/creatinine ratio and linear regression
parameters between 1,25-(OH)2-vitamin D3 value and albumin/creatinine ratio
Paramter

CoefficientStandard
error

Lower
confidence interval

Upperconfidence
intervalt Static

p-value

Uric acid

20.7728

8.7136

3.597837.9478

2.38400.0180

1,25-(OH)2vitamin D3
value-1.2347

0.8146-2.8403

0.3710-1.5156

0.1311

Correlation between 25-(OH)2-vitamin D3 status andalbumin/creatine ratio after adjustment based on multiple
factors
A positive association has been found has been foundbetween 25-(OH)2-vitamin D3 deficiency and albumin/creatine
ratio.

26

Table 3. Correlation between 25-(OH)2-vitamin D3 status andalbumin/creatine ratio after adjustment for multiple factors bymultiple linear regression
Parameter

CoefficientStandard
error

Lower
confidence interval

Upperconfidence
intervalt Static

p-value

Intercept-1.16310.4732-2.0960-0.2302-2.4582

0.0148Age

0.00570.0048-0.0038

0.0152

1.17900.2398Sex-0.0583

0.0817-0.2195

0.1028-0.7133

0.4765HbA1c

0.06300.02320.01730.10872.71680.0072BMI

0.00300.0093-0.0153

0.02130.32530.7453SBP

0.00500.00230.00050.00952.17450.0308Uric acid

0.04070.0265-0.0116

0.09301.53390.1266

Diabetes
duration-0.0015

0.0068-0.01480.0119-0.21460.8303Cholesterol-0.00080.0046-0.0100

0.0083-0.1758

0.8606

LDL-cholesterol-0.0005

0.0047-0.0097

0.0087-0.11380.9095

Tryglicerides

0.00010.0006-0.00100.00130.24150.8094HDL-cholesterol

0.00220.0052-0.00810.01240.41580.6780

1,25-(OH)2vitamin D3
deficiency

0.16490.07390.01930.31062.23250.0267

Our results are in concordance with the results from literaturedemonstrating that hyperuricemia and vitamin D deficiency playa role in the pathogenesis of diabetic kidney disease. It has
been demonstrated that uric acid increases oxidative stress andpromotes the activation of the renin-angiotensin-aldosterone
system (13, 14). Uric acid is associated with early diabetic
kidney disease (15, 16), and can be a factor that can predictthe progression of microalbuminuria (17). Observational studies
have demonstrated that serum uric acid and microalbuminuria
levels were significantly positively correlated with kidney diseasein patients with type 2 diabetes (18). Patients with higher uricacid levels have worse renal function, regardless of glycatedhaemoglobin (HbA1c) or the duration of diabetes mellitus(19). Xanthine oxidase (XO) is a very important enzyme thathas the role for converting sulfhydryl groups to uric acid. Data
from literature has demonstrated that an increase in uric acidby 1 μmol/L increased the risk of albuminuria by 1.5%, and anincrease in XO activity by 1 U/L increased the risk of albuminuria
by 1.5%. In diabetes mellitus patients, both XO and uric acid are
independently associated with albuminuria (20).
Numerous data support the association between low
1,25-(OH)2-vitamin D3 serum values and worse renal function.
In a study done on 1,216 patients with diabetes was identified
an independent association between vitamin D deficiency andthe onset of diabetic kidney disease (21). Levin and colleagues
Fiziologia - Physiology  2021.31.1 (101)demonstrated that in diabetes mellitus patients low serum levelsof 1,25-(OH)2-vitamin D3 were independently associated withlower glomerular filtration rate and with a high urinary albumincreatinine ratio (22). Also, supplementation with vitamin D can be
helpful in reducing the mortality of diabetes mellitus patients withvery advanced kidney disease, in a study done on haemodialysis
patients it was found that patients who received vitamin D
supplements had a significantly higher 2-year survival rate thanpatients who did not receive (23), suggesting efficacy. vitamin
D in reducing mortality in patients with end-stage renal disease.
Furthermore, animal studies have shown that serum levels of 25(OH) vitamin D and 1.25 (OH) 2 vitamin D3 were significantly
reduced in rats with diabetic kidney disease and treatment with
rat vitamin D effectively inhibited proliferation of mesangial cells
induced by hyperglycaemia (24). In another animal study, diabetic
mice with vitamin D -/- receptor mutations had more severe renal
impairment and were more likely to have severe proteinuria andglomerulosclerosis compared with wild-type mice (25).

4. CONCLUSION

In our study we demonstrated a statistically significant
correlation between uric acid levels and albumin/creatinine
ratio in univariate regression and between 25-(OH)2-vitamin
D3 deficiency and albumin/creatinine ration in multivariate
regression. The role of hyperuricemia and 1,25-(OH)2 vitamin
D3 need to be further explored for a better understanding of thepathogenesis of diabetic kidney disease.
Funding: none.
Conflict of interest statement: none declared.

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EXPLORAREA ASOCIERII ÎNTRE FACTORII DE RISC
CUNOSCUȚI ȘI EMERGENȚI PENTRU BOALA RENALADIABETICĂ ȘI RESPECTIV ALBUMINURIE LA PACIENȚIICU DIABET ZAHARAT DE TIP 2
RezumatPrezența albuminuriei la pacienții cu diabet zaharat tip 2 este un marker al disfuncției endoteliale și totodată unul dintre criteriile
diagnosticului bolii renale diabetice. Studiul de față a avut ca scop identificarea asocierilor între factorii de risc cardiovascular și
excreția renală de albumină la un grup de 218 pacienți cu diabet zaharat de tip 2. Valorile HbA1c, tensiunii arteriale sistolice, tensiunii
arteriale diastolice au fost semnificativ statistic mai mari la pacienții cu microalbuinurie sau macroalbuminurie comparativ cu pacienții cu
normoalbuminurie (p<0.01). Am identificat o asociere pozitiva statistic semnificativă între valorile acidului uric și albuminurie respectiv
deficiența de 25-(OH)2 vitamina D3 și microalbuminurie (p<0.01).
Cuvinte cheie: diabet zaharat, microalbuminurie, hiperuricemie, vitamina D

28

Fiziologia - Physiology  2021.31.1 (101)NECROTIZING FASCIITIS IN A PATIENT WITH HEPATIC
ENCEPHALOPATHY. CASE REPORT AND LITERATURE REVIEWDIANA MARIA CHIOREAN1,2*, MONICA DANIELA COTARCĂ4,5, BEATRICE BARAC6,RALUCA NICULESCU1,2,3, MIHAELA CORNELIA ȘINCU1,2, ANDREEA CĂTĂLINA
TINCA1,2,3, IULIU GABRIEL COCUZ1,2,3, ADRIAN HORAȚIU SABĂU1,2,3, PAUL ANTONIUBOȚAN7, OVIDIU SIMION COTOI2,3
1

Doctoral School of Medicine and Pharmacy, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Târgu Mureș, 38
Gheorghe Marinescu Street, 540142, Târgu Mureș, România

2

Department of Pathology, County Clinical Hospital of Târgu Mureș, 6 Bernády György Square, 540072, Târgu Mureș, România

3

Department of Pathophysiology, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Târgu Mureș, 38 Gheorghe
Marinescu Street, 540142, Târgu Mureș, România

4

Department of Pathophysiology, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square, 300041, Timișoara, România

5

OncoGen Center, Pius Brânzeu Clinical Emergency Hospital, 156 Liviu Rebreanu Boulevard, 300723, Timișoara, România;6
Department of Pharmacology, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square, 300041, Timișoara, România

7

Department of Plastic Surgery, County Clinical Hospital of Târgu Mureș, 6 Bernády György Square, 540072, Târgu Mureș, România
ABSTRACTIntroduction: Necrotizing fasciitis, considered to be a very rare and severe soft tissue infection, is associated with rapid progressive necrosisof subcutaneous tissue and superficial fascia. Although this rare condition has a very high mortality rate and requires prompt diagnosis as
well as urgent treatment, there are no defined blood tests that can diagnose it. Case presentation: In this report, we present the case of a51-year-old patient, known with mental and behavioral disorders due to alcohol abuse, liver cirrhosis, early liver encephalopathy, anemia,
and hypoproteinemia, who presented to the Dermatovenerology department with a main complaint of hematoma at the level of the jointof elbow, appeared as a result of physical aggression. Despite the medical care, the patient’s situation deteriorated, more accurately
with the extended progression of erythema and sorrow, with deep hyperpigmentation and extended tenderness. As a consequence, thepatient was transferred urgently to the plastic surgery department with the suspicion of necrotizing fasciitis as a diagnosis. Conclusion:
Knowing that soft tissue necrotizing infections have an acute, aggressive, and rapidly progressive character, in patients with cirrhosis theyoccur more often than in the general population, because the phenomenon of bacterial translocation occurs in the intestinal lumen as aconsequence of intestinal bacterial growth, increased permeability and decreased motility. Excessive growth of intestinal bacteria is also
responsible for the occurrence of hyperammonemia, leading, consequently, to the appearance of hepatic encephalopathy. Considering
those presented, complications place this group of patients in the category of those at high risk of mortality.
Keywords: fasciitis, cirrhosis, encephalopathy, necrosis, bacterial translocation

INTRODUCTION

Necrotizing fasciitis is considered to be a very rare andsevere soft tissue infection, associated with rapid progressive
necrosis of subcutaneous tissue and superficial fascia. Although
this rare condition has a very high mortality rate and requires
prompt diagnosis as well as urgent treatment, there are no
defined blood tests that can diagnose it. At the onset, it is difficult
to differentiate from other superficial skin conditions, so early
detection and surgical debridement, as well as an aggressive
antimicrobial therapy, are essential to limit the loss of limb orlife [1]. Clinical features of necrotizing fasciitis include severe

regional cellulitis with ill-defined margins, soft-tissue edema withgrayish-brown discharge, severe pain, skin necrosis with bullae
formation (blisters containing serous fluid), crepitus (grating
sound), and sloughing of the skin followed by numbness of theinvolved region due to destruction of nerves in the fascial planes[17,18]. The bacteria can also result in ischemia of the involved
region due to thrombosis of blood vessels which eventually
results in gangrene. Limbs are the most common site involvedin this life-threatening disease, followed by the perineum andtrunk [17,19].

Received 5th of June 2021. Accepted 7h of July 2021. Chiorean Diana Maria, County Clinical Hospital of Târgu Mureș, 6 Bernády György Square, 540072,
Târgu Mureș, Mureș, România. Telephone: +40760513978. E-mail: chioreandianamaria@yahoo.com

2021.31.1 (101 )  Fiziologia - Physiology

29

CASE PRESENTATION

We present the case of a 51-year-old male patient, knownwith mental and behavioral disorders, hepatic cirrhosis, and early
hepatic encephalopathy due to alcohol abuse. He presented tothe dermatovenerology department with a main complaint ofhematoma at the level of the joint of the elbow, which appeared
as a result of physical aggression. At admission, the patient’s
psychopathological picture was dominated by psychomotor
anxiety, extremity tremor, nausea, vomiting, profuse sweating,toxicophilic behavior, overall decreased efficiency, and irritability.
Throughout the hospital stay, a diagnostic of erysipelas has
been established and the patient was treated with five dosesof Metamizole sodium monohydrate 1g/2ml intravenous (i.v.),
two doses of Alprazolam 1mg i.v., ten doses of Ceftriaxone
1g i.v., one dose of Dexamethasone 8mg/2ml i.v., ten dosesof Gentamicin sulfate 40mg/ml i.v., ten doses of Omeprazole
20mg, 500 ml of lactated Ringer i.v., four doses of Diclofenac
sodium 75mg/ml i.v., fifty tubes of Gentamicin sulfate ointment
0,1% and two-hundred tubes of Diclofenac sodium gel 5%/1%
menthol. No blood tests or X-Rays were performed. Despite
the medical care, the patient’s situation deteriorated, moreaccurately with the extended progression of erythema andsorrow, with deep hyperpigmentation and extended tenderness.
As a consequence, the patient was transferred urgently to theplastic surgery department with the suspicion of necrotizing
fasciitis as a diagnosis.
On laboratory testing, the metabolic panel was notable for a
gamma-GT of 402 U/L, low density lipoprotein 170 mg/dL,
potassium 6,14 mmol/L, sodium 133 mmol/L, urea 72 mg/dL.
Complete blood count revealed the number of leukocytes as
30,86/µL, neutrophiles 26,18/µL, monocytes 1,30/µL, bazophils
0,44/µL, eritrocytes 3,88/µL, HEM 33,71 pg, VTM 11,18/fL, PCT
0,37%, neutrophiles 84,83%, limphocytes 9,09%.

The biochemistry part was notable for uric acid as 7,93 mg/dL
and alanine transaminase as 120 U/L.
Emergency debridement was performed (Figure 1). Under
troncular anesthesia, the incision and wide excision of the septic30

necrotic tissue were performed, which revealed subcutaneousedema. During the surgical procedure, a purulent material
was collected, which revealed a positive response for group A
beta-hemolytic streptococcus, penicillin-sensitive. The patient
was treated with Amoxicillin/ Clavulanic acid, Fluconazole,
Gentamicin, Metronidazole, lactated Ringer, Plasma volume
substituent 6%, Alprazolam, Omeprazole. The infection was
kept under control due to antibiotics and the patient’s status
improved. After the patient has been discharged, we had nolonger information regarding the evolution of his health condition.
We kept him under observation for a period of twenty-one days.
Fig. 1. Images of the emergency debridement

DISCUSSIONS

Even though necrotizing fasciitis has been a known clinicalelement for a considerable length of time, the term was firstutilized by Wilson in 1952 to portray „fascial corruption” [5]. As
referenced in the past lines, pathogens can spread quickly along
the fascial planes. When the microbes are particularly inoculated,
the toxins spread through the skin, from the epidermis to profoundmuscle tissue in a quickly spreading way, causing corruption of
the wide tissue and its destruction [10]. Therefore, the expression„time is fascia” [3] appears to be proper, which has led to thebuilt-up conviction that controlling sources through the early
medical procedure [6] to expel necrotic and tainted tissue candiminish the movement of the contamination and improve theresults [7]. Complications observed with a higher risk of death,
incorporate diabetes, stoutness, congestive cardiovascular
breakdown, fringe vascular ailment, constant liver malady, andmalignancies [14]. Alluding to our case, contaminations are basic
in patients with hepatic cirrhosis. They occur more regularly inthese categories of patients than worldwide. One possible reason
behind this is the bacterial translocation of the intestinal lumen
that occurs due to intestinal bacterial abundance, extended
permeability, and decreased motility [2]. The abundance ofintestinal bacteria is also responsible for hyperammonemia,
causing hepatic encephalopathy [9]. Patients with cirrhosis andnecrotizing fasciitis require long-term hospitalization and chargea large number of hospital fees, making necrotizing fasciitis the
hospital’s most expensive diagnosis [1]. Direct inoculation of
adjacent necrotic tissue or spread in the lymphatic tract canlead to the establishment of extremity infection and is therefore
considered a fatal skin disease [11]. According to reports, its
mortality rate is very high, about 4.8 per 1,000,000 years perperson [8]. It can affect all age groups, especially older men,with a total death rate of almost 30%, which will increase at
extreme ages. Regarding treatment, one with 100% oxygen
at atmospheric pressure of two to three times can improve
the oxidative explosion of leukocytes, limit the development ofanaerobes and restrict the arrival of bacterial poisons, however,it should not delay the medical crisis, procedure, and strong fluid
invigoration [12].

PATHOLOGICAL ASPECTS
Fiziologia - Physiology  2021.31.1 (101)Necrotizing fasciitis can progress in two manners:
1. The penetrant manner, where organisms or the spores get
inoculated via a break in the integrity of skin or mucosa;
2. The nonpenetrant manner, where a deep tissue injury inindividuals with transient bacteremia leads to an influx oforganisms that gain access via deep tissue vessels to thesurrounding tissues.
In both manners of the entrance, microbes once inoculated,
release exotoxins altering host repair mechanisms leading toleucocyte–platelet aggregation resulting in widespread tissue
damage, occlusion of vessels causing tissue necrosis, andexaggerated inflammatory responses [4],[13]. Invasion of thesemicrobes is recognized by the host immune system leading toa widespread influx of acute inflammatory cells with an aim
to kill these pathogens [4]. Regarding the type of infection,these can be monomicrobial or polymicrobial, with causative
agents as anaerobes (Clostridium perfringens, Bacteroides
fragilis), Streptococcus species (including group A betahemolytic streptococci), and Staphylococcus species. Group A
Streptococcus is well established as the most common pathogen
causing necrotizing fasciitis [16]. This kind of bacteria can cause
a wide variety of infections that range from minor illnesses,
such as strep throat and mild skin infection, to very severe andlife-threatening post-streptococcal diseases, such as rheumatic
fever and glomerulonephritis [17].
From a histopathological point of view, as in the case of ourpatient, the microscopically evaluated skin fragments wereinterested in an extensive process of deep necrosis of theepidermis, dermis, and even subcutaneous tissue. A moderate
polymorphic inflammatory infiltrate consisting of granulocytes
and lymphocytes was identified. Rare plasma cells and rareeosinophils have also been observed (Figure 2).

Fig. 2. Images of the microscopic perspective – hematoxilin andeosine, 10 x magnification

CONCLUSION

Our case raises the possibility that cirrhosis could lead tonecrotizing fasciitis because of the simultaneous presence of2021.31.1 (101 )  Fiziologia - Physiology

additional predisposing factors. Although a definitive connection
cannot be made based on our case alone, we suggest further
investigations regarding this aspect.
Acknowledgments: Conflict of interest – none to declare. The
article is an original work and the final form was not published
before.

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9. Oud L, Watkins P. Necrotizing Fasciitis Among Patients With
Liver Cirrhosis in Texas, 2001 - 2010: A Population-Based Cohort Study.
J Clin Med Res. 2016 Feb;8(2):90-6. doi: 10.14740/jocmr2420w. Epub2015 Dec 28. PMID: 26767076; PMCID: PMC4701063.
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Susceptibility of microorganisms causing acute hand infections. PLoS
One. 2019 Aug 22;14(8):e0220555. doi: 10.1371/journal.pone.0220555.
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11. Rigopoulos N, Dailiana ZH, Varitimidis S, Malizos KN. Closedspace hand infections: diagnostic and treatment considerations. Orthop
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necrotizing soft-tissue infections. Infection. 2019 Aug;47(4):677-679. doi:
10.1007/s15010-019-01316-3. Epub 2019 May 10. PMID: 31077024.
13. Stevens DL, Bryant AE. Necrotizing Soft-Tissue Infections.
N Engl J Med. 2017 Dec 7;377(23):2253-2265. doi: 10.1056/
NEJMra1600673. PMID: 29211672.
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14. Al-Qurayshi Z, Nichols RL, Killackey MT, Kandil E. Mortality Risk
in Necrotizing Fasciitis: National Prevalence, Trend, and Burden. Surg
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17. Hill HR. Fitness genes of group A streptococci in necrotizing
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10.1172/JCI126482. Epub 2019 Jan 22. PMID: 30667379; PMCID:
PMC6355235

FA S C E I TA N E C R O T I Z A N T Ă L A U N PA C I E N T C U
ENCEFALOPATIE HEPATICĂ. PREZENTARE DE CAZ ȘI
ANALIZA LITERATURII DE SPECIALITATEREZUMAT
Introducere: Fasceita necrotizantă, considerată a fi o infecție foarte rară și severă a țesuturilor moi, este asociată cu necroza rapid
progresivă a țesutului subcutanat și a fasciei superficiale. Deși această afecțiune rară are o rată de mortalitate foarte ridicată și necesită
un diagnostic prompt precum și un tratament urgent, nu există teste sanguine specifice care să o poată diagnostica. Prezentare decaz: În acest raport, prezentăm cazul unui pacient în vârstă de 51 de ani, cunoscut cu tulburări mentale și comportamentale datorate
abuzului de alcool, cirozei hepatice, encefalopatiei hepatice precoce, anemiei și hipoproteinemiei, care s-a prezentat la departamentul
de dermatovenerologie a clinicii noastre cu drept prinicipală acuză a unui hematom la nivelul articulației cotului, survenit în urma unei
agresiuni fizice. În ciuda îngrijirilor medicale, statusul pacientului s-a deteriorat semnificativ, cu extinderea semnificativă a eritemului, ohiperpigmentare profundă și extinderea sensibilității. În consecință, pacientul a fost transferat în regim de urgență la departamentul dechirurgie plastică, cu suspiciunea de fasceită necrozantă, drept diagnostic.
Concluzie: Cunoscând faptul că infecțiile necrotizante ale țesuturilor moi au un caracter acut, agresiv și rapid progresiv, în cazulpacienților cu ciroză hepatică acestea apar mai des decât în ​​cadrul populației generale, datorită fenomenului de translocație bacteriană
de la nivelul lumenului intestinal - consecință a creșterii bacteriemiei, a permeabilității crescute și a scăderii motilității intestinale. Excesul
bacteriilor intestinale este, de asemenea, responsabil pentru apariția hiperamonemiei, ducând, în consecință, la apariția encefalopatiei
hepatice. Ținând cont de toate acestea, complicațiile apărute fac ca acest grup de pacienți să prezinte un risc crescut de mortalitate.
Cuvinte-cheie: fasceită, ciroză, encefalopatie, necroză, translocație bacteriană

32

Fiziologia - Physiology  2021.31.1 (101)THE GENETIC AND ACQUIRED HOST SUSCEPTIBILITY TO
NON-TUBERCULOUS MYCOBACTERIAL INFECTIONSBICIUSCA VIOREL, ⃰2 SORINA IONELIA STAN, ⃰3 PATRICIA DURAND, #4 MARABALTEANU,#5 BICIUSCA TEODORA, 6 BEATRICE MAHLER⃰1

Department of Internal Medicine, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania.
Department of Internal Medicine, Resident Ph, Emergency County Clinical Hospital Craiova, Romania.
3
Department of Internal Medicine, Resident Ph, ”Filantropia”Municipal Clinical Hospital Craiova, Romania.
4
Department of Pneumology, Faculty of Medicine, ”TituMaiorescu” University, ”Prof. Dr. Marius Nasta” Institute for Pneumology, Romania.
5
Department of Radiology, ”Goethe” University, Faculty of Medicine, Frankfurt am Main, Germany.
6
Department of Pneumology, Faculty of Medicine,”Carol Davila” University of Medicine and Pharmacy Bucharest, ”Prof dr Marius Nasta” Institute forPneumology, Romania.
⃰These authors have equal contribution
1
2

ABSTRACT
Non-tuberculous mycobacteria (NTMs) are ubiquitous, non-pathogenic microorganisms that produce lung/ pulmonary, extrapulmonary
and disseminated infections in people with immune deficiencies. If lung diseases with NTM (LD-NTM) occur mainly in immunocompetent
individuals with pre-existing respiratory diseases, extrapulmonary and disseminated infections occur only in immunocompromised hosts.
Both local risk factors and systemic risk factors are of particular importance in the pathogenesis of NTM infection. Genetic susceptibility
plays an important role in the pathogenesis of extrapulmonary infections and disseminated with NTM, while acquired susceptibility plays
an important role in lung infections, but also extrapulmonary and disseminated with NTM. Recognition of these susceptibilities has adecisive role both in establishing the diagnosis of NTM infection and in appreciation of appropriate therapeutic conduct, especially inpatients with genetic susceptibility.
Key words: NTMs, genetic and acquired host susceptibility, host risk factors

INTRODUCTION

The incidence of NTM infection is increasing worldwide[1]. The increase in life expectancy and the higher incidenceof this infection in the elderly segment of the population
have fully contributed to this fact [2]. Also, the number of
immunosuppressed patients of all ages is constantly increasing
due to the increased incidence of HIV/ AIDS, but especially due
to medical advances in treating various autoimmune diseases
and the availability of organ transplants as a method of treating
various serious diseases [3].A step forward in the managementof these infections was the improvement of diagnostic techniques
for NTM infection [4]. On the other hand, the diagnostic
difficulties generated by the colonization with NTM of various
biological samples and the total ignorance of the predisposing
factors of these infections led to a more difficult managementof this disease [5]. In addition, given the difficulty of eradicating
NTM and its considerable recurrence, the identification ofsuitable candidates for treatment and the timing of initiation oftherapy are also challenging [6]. Given that, in many developed
countries, the incidence of NTM infection exceeded theincidence of tuberculosis, it is necessary to better understandthe pathogenesis of this disease, namely to understand which
are the population risk groups that develop these infections [7].

If NTM-LD occurs predominantly, in patients with pre-existingorganic lung disease (COPD, emphysema, pulmonary fibrosis,
bronchiectasis), extrapulmonary damage and the disseminatedform of NTM infection most often occur in immunosuppressedindividuals, through genetic or acquired causes [8].
This review summarizes the main risk factors for lung anddisseminated NTM infection and indicates a strategy for
identifying genetic and acquired immunodeficiencies.

GENETIC SUSCEPTIBILITY

Human leukocyte antigen alleles. Although the suspicion
that tuberculosis is a hereditary disease existed before thediscovery of the tubercle bacillus in 1882, with the discoveryof the pathogen of tuberculosis, the importance of genetic
susceptibility to this disease was largely ignored [9]. It was not
until the last decades of the twentieth century that scientificevidence of the genetic predisposition to bacillary infection began
to appear. The relationship between tuberculosis infection and
HLA-DR2 histocompatibility antigens has been known for over
30 years [10]. There are authors who have established a link
between innate susceptibility to mycobacterial infection andcertain human leukocyte antigens [11], mainly those of class Iof antigens A10 and B8 and of class II DR2 (DRB1 1501)[2]. For

Received 24th of June 2021. Accepted 19th of July 2021. Address for correspondence: Balteanu Mara, PhD, ”Titu Maiorescu” University, Faculty of Medicine,
Department of Pneumology, Gheorghe Petrascu Street no 67A, RO-031593 Bucharest, Romania, phone:+40213243013, email: mara.balteanu@prof.utm.
ro; BiciuscaTeodora, PhD student, Goethe University Frankfurt, Theodor W Adorno Platz 1, 60323 Frankfurt am Main, Germany, phone:+4915202839885,
email: teodorabiciusca96@gmail.com.

2021.31.1 (101 )  Fiziologia - Physiology

33the first time, the involvement of the genetic factor in the spreadof MAC infection in HIV-positive patients was demonstrated by
Naik, who showed an association with the alleles DR2, DRB1
0601 and a negative association with the alleles DMA 0102 [12].
Subsequently, the association between the polymorphism of theclassic HLA class II genes (DRB1 and DQB1) and the occurrenceof disseminated MAC infection in HIV-1 positive patients was
extended to the DMA and DMB accessory genes. In anothermulticenter study, it was shown that not only the polymorphismof genes encoding DRB1 alleles, but especially the presence ofallelic combinations between DRB1 and DMA and DMB, plays animportant role in the occurrence of MAC-disseminated disease
in HIV-1 patients [13]. On the other hand, two Japanese study
groups showed the presence of HLA antigens DR6, Dr4, A33
and A26, as common among patients with LD-MAC without preexisting lung disease [14,15]. Also, the presence of HLA-A26 has
been associated with disease progression.
Mendelian susceptibility to mycobacterial diseases(MSMD) is an immunodeficiency syndrome of genetic cause,with increased susceptibility to pathogenic and opportunistic
mycobacteria in the environment [16].Only half of patients with
MSMD have an identified genetic cause [17]. To date, nine
genes responsible for MSMD are known [18]. Seven of these
are inherited in an autosomal recessive or autosomal dominantmodel (Ƴ-IFNR1, Ƴ-IFNR 2, STAT1, IL12B, IL12RB1, IRF8 andI SG15 genes) and two are related to X (IKBKG and CYBB
genes) [1]. In these patients, BCG vaccination causes a systemic
and severe form of mycobacterial disease, often fatal, which
is why BCG vaccination should be avoided. The most serious
forms of the disease are autosomal recessive deficiencies ofcomplete interferon gamma 1 (IFN-gammaR1) and receptor
2 (IFN-gammaR2)[19]. MSMD due to 1-IFN receptor 1 and 2
deficiency is a genetic condition with autosomal recessive andautosomal dominant transmission. The autosomal recessive
form, characterized by complete receptor deficiency for Ƴ-IFN, ismanifested from childhood by the appearance of: disseminatedforms of mycobacterial infection after BCG vaccine, severe andrecurrent infections with mycobacteria or by the inability to form
well-circumscribed mycobacterial granulomas [20].The diagnosis
is suggested by the complete absence of receptor expression
on the cell surface. The therapy of these syndromes is basedon antimycobacterial treatment, because cytokine replacement
therapy (Ƴ-IFN) has no results [21]. The autosomal recessive
form, characterized by partial Ƴ-IFN receptor deficiency on thecell surface, is manifested later in childhood or adolescence by
localized or disseminated infections after BCG vaccination, as
well as by pulmonary or osteoarticular infections with NTM, butwith retained capacity of mycobacterial granuloma formation.
Multifocal osteomyelitis with NTM is a feature of these patients[22]. The diagnosis is suggested by a 3 - 5-fold increase in R1
receptor expression on the surface of monocytes. The diagnosis
is confirmed by sequencing the gene encoding the R1 receptorfor Ƴ-IFN. Mycobacterial infections should be treated aggressively
with antimycobacterial drugs, and some patients may also
respond to Ƴ-IFN[23].
34

MSMD due to deficiency of the p40 subunit of IL-12 occurs
as a result of a mutation in the gene encoding the IL-12B subunit.
This genetic condition, with autosomal recessive transmission,
is manifested by systemic infections after the administration of
the BCG vaccine, systemic infections with NTM and sepsis with
Salmonella [24]. These infections can occur from childhood, but
can also manifest later with localized mycobacterial infections.
The diagnosis is suggested by the absence of secretion of
the p40 subunit of IL-12. These infections respond favorably
to the administration of antimycobacterial drugs, but also to
the additional administration of cytokines (Ƴ-IFN), when thetherapeutic response to antimycobacterial therapies partial [25].
MSMD due to IL-12 β1 receptor deficiency is an autosomal
recessive genetic disorder. Lack of expression of these receptors
has the effect of decreasing gIFN secretion from lymphocytes
and NK cells. This genetic condition is manifested by extent
infections with NTM. After BCG vaccination, patients withthese genetic defects may develop a systemic infection, andthe developed tissue granulomas are well organized [26]. The
diagnosis is suggested by the absence of IL-12 receptors on thesurface of T lymphocytes and deficient STAT4 phosphorylationin response to IL-12 lymphocyte stimulation [27].Treatmentof NTM infections in these patients should be performed with
antimycobacterial antibiotics and cytokines (Ƴ-IFN), as the gIFN
receptor and downstream stimulation are intact [28].
MSMD due to STAT1 deficiency is a genetic disorder with
autosomal dominant and autosomal recessive transmission,
which occurs as a result of a mutation in the gene encoding
STA1 synthesis, which is a key translator of the IFN type II(Ƴ-IFN) signal and IFN type I (α/β-IFN)[29].The autosomal
dominant form, characterized by altered STAT1 dimer formation
following Ƴ-IFN induction, results in a defective Ƴ-IFN signaling,
but with an intact α/β-IFN-mediated antiviral defense. Patientswith this genetic defect who survived mycobacterial infections
in childhood have reached adulthood. The autosomal recessive
form (homozygous), characterized by altered signaling in both
IFN type I and IFN type II, is manifested by mycobacterial
infections from the first year of life, fatal, these patients being
ideal candidates for stem cell transplantation [30].
Heterozygous mutations of the cystic fibrosis
transmembrane conductance regulator (CFTR) genemanifests phenotypically through CF manifestations such as:
chronic pancreatitis, sinusitis and bronchiectasis [31] and over
25% of the patients with CF have evidence of NTM infection(LD-NTM) [32]. In case of CF patients isolated NTM lung disease(NTM-LD) occurs more often. In CF patients the species varies
between geographical regions with M.avium complex (MAC), M.
kansasii and M.abcessus being the most common in the UnitedStates and M.abcessus being the most frequent in Europe [33].
There is also a reported transmissibility of M.abcessus from one
patient to antother. A study by Kim showed that patients with asingle mutation of CFTR have greater progression of radiologicallesions [33], while other authors have shown that the nodularbronchial form appears in those patients with CF effusion[34].
ZIedalski evaluated 50 patients with idiopathic bronchiectasis
Fiziologia - Physiology  2021.31.1 (101)or NTM-LD (20 patients) for CFTR mutations and performed
iontophoresis, resulting 50% being homo or heterozygous
for the CFTR gene and 20% met the HR criteria[35]. A 24%
incidence of at least one CFTR gene mutation in the patientswith NTM-LD was showed in another report [36]. Jang et al[37] investigated the relationship between CFTR mutations andthe susceptibility to NTM lung infection in a Korean population,
showing that CFTR genes variants can increase risk to NTM
infections and Q1352H mutation is significantly higher in in
NTM patients in the Korean population. Mutations in the CFTR
gene are more common in Europeans [38], and tend to become
more frequent as other rare alleles of CFTR are added to themore than 1500 mutations already known [39]. In a study by
Bar-On to asses NTM prevalence in CF patients, M.abcessus
was the most prevalent NTM and caused prolonged infection
despite therapy [40].
There are few articles on the association between mutations in
the gene encoding α-1 antitrypsin (AAT) and NTM-LD. Somephenotypic conditions predispose to NTM lung disease, suchas emphysema and bronchiectasis. AAT prevents emphysema,
bronchiectasis and protects the host against NTM infection.
Also it enhances macrophage immunity against NTM [41].
Heterozygous AAT mutations are more often found in patientswith NTM-LD compared to general population [42]. Kim andcolleagues compared the tomographic aspects of NTM-LD inpatients with normal and pathological phenotypes of AAT andfound no differences in the diagnosis or follow-up [3]. Kaminska
et al. studied patients with bronchiectasis and lung infections
with rapidly growing NTM (RG-NTM) resulting that 27% had
anomalous AAT proteins while M.abcessus was isolated in
64% of patients [43]. It remains unclear whether AAT mutations
predispose to NTM- LD- independent of other respiratory
diseases associated with AAT deficiency [44].
Natural resistance-associated macrophage protein
1 (NRAMP1) or SLC11A1 is an iron ion transporter that is
located in the lysosomal membrane during phagocytosis[45] with evidence from the literature data for an association
between some polymorphisms of SLC11A1 and NTM-LD. Koh
et al [46]compared the prevalence of three polymorphisms of
the SCL11A1 gene in patients with type 2 NTM-LD (MAC and
M. abcessus) vs control subjects resulting that heterogeneityof some exon regions of codon 543 has been associatedwith an increased risk of NTM-LD. However, initial studies
by Huang et al [47] found no associations between SCL11A1
gene polymorphism and NTM-LD and a meta-analysis foundno association between SCL11A1 and tuberculosis (TB) [48].
Sapkota et al [49] investigated the genomic structure of SLC11A1
and the association with MAC infection in a Japanese populationand observed that rs2279014 T allele is an SNP exhibiting
protection against the infection.
Vitamin D has well-expressed receptors on the surface oflymphocytes and macrophages that exert immunomodulatory
effects, by suppressing the production of ϒ-IFN and IL-12 that
promote the proliferation and activation of Th2 lymphocytes
more than that of LTh1[50]. LTh1 is currently thought to2021.31.1 (101 )  Fiziologia - Physiology

produce cytokines that promote granuloma formation andmediate cellular immunity, while LTh2 mediates humoral
immunity[51]. The imbalance between Th1 and Th2 responses
has been described in many immunological and infectious
diseases, including diseases such as asthma [52,53], atopy[2], ulcerative colitis[54], pulmonary tuberculosis [55], leprosy
and leishmaniasis [56]. Starting from the finding that vitamin D
receptor (VDR) polymorphism is associated with the risk of TB[57], it has been shown that the polymorphism of this receptor canalso be associated with NTM-LD susceptibility. The VDR gene islocated on chromosome 12q13 and has 4 classical nucleotide
polymorphisms, FolkI, BsmI, ApaI and TaqI, which have been
extensively studied and have been reported to affect risk invarious pathologies. The FokI site is a functional polymorphismof the VDR gene that can alter the amount of VDR produced
and correlates with the plasma levels of vitamin D in patientswith TB [58]. Numerous epidemiological studies have suggested
that low levels of vitamin D in the host are associated withincreased susceptibility to TB [59,60,61]. Vitamin D deficiency
is also associated with extrapulmonary dissemination in TB,
which indicates that vitamin D status is associated with thedisease phenotype [62]. In this regard, Geder et al. [63] tested
56 patients with M.malmoese lung disease for three types ofreceptor polymorphisms and identified a lower prevalenceof Flavobacteriumokeanokoites I (FokI) polymorphism while
the prevalence of Acinetobacterpasteuranis, subspecies IA
and Thermusaquaticus YTI (TaqI) subspecies was increased.
However, Tanaka et al [64] did not find any difference for FokI and
TaqI polymorphisms in sick people compared to healthy controls.
The 2 toll-like receptor (TLR2) has the role of mediating
the recognition of bacterial antigens by the innate immune
system [65]. This receptor is recognized as the receptor bywhich macrophages recognize mycobacteria [66]. A study by
Ryu et al. [67] which included 17 patients with NTM-LD (MAC, M.
abscenssus), measured mRNA production for TLR2, TNF, Il-12,
α-TNF and the p40 subunit of Il-12 in blood monocytes. The levelof these substances after exposure to MAC was lower comparedto the level produced by monocytes in healthy subjects. Thus,
exposure to MAC in the presence of anti-TLR2 antibodies
reduces the production of mRNA and cytokines in controls
compared to patients with NTM, suggesting a TLR2 defectthat confers NTM susceptibility to patients. TLR2 is present inmacrophages, dendritic cells and lung epithelial cells. Expressionof phosphatidyl-myo-inositol surface molecules (PIMS) on
M.abscessus stimulates the immune response of macrophages
via TLR2 [68]. Stimulation of TLR2 also results in the releaseof IL-8 [69]. Certain variants of M. abscessus also stimulate therelease of TNF-α following interaction with TLR2 [70].

ACQUIRED SUSCEPTIBILITY. OBSTRUCTIVE
PULMONARY DISEASES (ASTHMA, COPD)

Local conditions in obstructive pulmonary diseases like
COPD and asthma, such as impaired clearance of secretions,abnormal composition of airways surface liquid, abnormal
35

composition of sputum and airway damage caused by persistent
inflammation could increase the risk for association with NTM[71]. In COPD, parenchymal destruction may be the factor that
impairs clearance of inhaled pathogen. In asthmatic patients,
bronchial hyperresponsiveness may impair mucociliary clearance[72]. In 2016, Marras and al [72] described a high association
between obstructive diseases and NTM pulmonary diseasein an over 6 million people study (COPD and asthma wereassociated with approximately nine-fold and five-fold higher
adjusted incidences of NTM-LD). The same high association
rates were found in 2013 by Andrejak and al.[73] in a study on
332 Danish patients with NTM infection. The risk infection rate inthese subjects had an odd ratio (OR) of 15.7 (95% CI 5.2–11.6)
for NTM infection among patients with COPD, a higher OR of
29.1 for COPD patients receiving inhaled corticosteroid therapy
and an OR of 7.8 in asthma patients, compared to the general
population. A recent study [73] revealed that NTM pulmonary
disease is associated with nodular/bronchiectatic changes onchest CT in 47% of cases and with the form of infiltrative/cavitary
in 35% of cases. According to microbiological data in the same
study, M. avium was present in 39% of the examined population,
the most frequently strain associated with obstructive pulmonary
diseases. M. gordonae is the only strain without association with
NTM lung diseases. Marras and al [72] described the presenceof M. xenopi and M. avium complex (MAC) and founded higher
rates of cavitation in patients with M.xenopi than at patients with
MAC (46% vs. 16%; p = 0.01). M. xenopi was associated with
COPD and death risk. The high mortality risk for patients with
COPD and NTM infection was found by Pyarali and al [74] tobe 1.43 times higher compared to uninfected COPD patients.
There are studies [75] that described the association between
inhaled corticosteroid use and risk of pulmonary NTM, taking inaccount that the local immunity may be compromised by ICS
therapy. There is risk associated with dose and type of inhaledcorticosteroid and also with the systemic corticosteroid use [76].
Balavoine and al [77] underlies the risk of association between
chronic macrolide treatment in COPD and the selection of theresistant NTM.

BRONCHIECTASISThe pulmonary infections of non-CF bronchiectasis due
to NTM are increasing worldwide. In a study performed by
Mirsaeidi et al on 182 patients with non-CF bronchiectasis andNTM lung infection, NTM was isolated in 68 (37%) of patientsand the most common was M. avium complex (MAC) [78]. In a
study performed by Koh WJ et al to determine the frequency of
NTM lung infection in patients with bilateral bronchiectasis andbronchiolitis in CT findings, NTM lung infection was isolated in 36(34%) of 105 patients, with the most common isolated organism
being MAC in 50% of the patients [79]. One pathophysiologicalmechanism involved in NTM lung infection in the patients withbronchiectasis is the adhesion of the NTM to the damaged areasof the respiratory mucosa. Fibronectin attachment protein from
NTM binds to the one from the extracellular matrix from thedamaged mucosal surface [80]. The main clinical symptoms
36

for the non-FC patients with bronchiectasis and NTM infection
are cough, sputum production, wheezing and dyspnea. There
are also some phenotypic features that are associated with
NTM infection: the female gender, hence low body mass index(BMI), pectus excavatum, scoliosis and mitral valve prolapse[81]. The treatment of patients with non-CF bronchiectasis andNTM lung infection includes management of bronchiectasis withhypertonic saline or positive pressure devices in stable patientsand management of the NTM lung infection [81].
Gastroesophageal reflux disease (GERD)
GERD is characterized by reflux of gastric contents, whichcauses various symptoms and respiratory complications.
It is associated with many lung diseases, such as asthma,
bronchiectasis, chronic bronchitis but also with NTM infection
with the appearance of a lesion in the bronchial epithelium
secondary to the reflux of gastric contents [82]. The association
between NTM-LD and GERD has been reported by many
authors, so Hadjiliadis [83] and Griffith [84] reported an increased
prevalence of M. fortuitum and M. abscessus infection inthese patients. Numerous reports have stated that GERD is apredisposing factor for NTM lung disease. However, insufficient
data is available on treatment with proton pump inhibitors thatsuppress acid secretion and thus predispose to NTM or represent
a marker of severe GERD [85].

DIABETES MELLITUS

Diabetus mellitus is a metabolic disease with countless
manifestations and complications, including infections caused
by NTM, which appear as complications in these patients due tothe lack of clear diagnostic methods[86]. The most associatedlocalized infections with NTM are: cutaneous produced byM.chelonae [87,88] at the site of insulin injection, pleural with
MAC [89], pulmonary and musculoskeletal with M. Kansassii[90], thyroid with MAC [91], but without a clear incidence of
infections in diabetic patients to date. Patients with diabetes arevulnerable to NTM infections, due to the resistance of infections
to antituberculosis treatments and routine diagnostic methods
are required. Early treatment will lead to a rapid improvement but
also an assessment of the susceptibility of antibiotic treatment
to these microorganisms [88].

CHRONIC KIDNEY DISEASE

Chronic kidney disease is a risk factor for NTM infections[92], associated with infections caused by cutaneous, peritoneal[93], bone, joint nontuberculous mycobacteria, especially slowgrowing mycobacteria [94,95]. Peritonitis secondary to peritoneal
dialysis is a major cause of morbidity and mortality in thesepatients [96]. Patients with kidney disease also have a risk ofinfection with NTM related to the source of contaminated water,
with a high risk of peritonitis. However, M. tuberculosis is themost common pathogen for peritonitis with negative cultures,
while the most common species of NTM that cause this typeof infection are M. Fortuitum (39%) and M. Chelonae (14%).
Both species are considered fast-growing mycobacteria, being
Fiziologia - Physiology  2021.31.1 (101)isolated seven days after growth [97],[98].
CANCER PATIENTSCancer patients are also at an increased risk of NTM
diseases on one hand because of the decreased cell-mediatedimmunity and on the other hand because of chemotherapy.
Patients with lung cancer have a particularly high risk ofdeveloping NTM infections, probably because of the damaged
airways, but hematological malignancies also represent anincreased risk of localized catheter-associated and disseminatedinfections. Hairy cell leukemia has been associated with NTM
infections since 1980 [100]. Other diseases that pose a potential
risk for NTM infections include granulomatous diseases, variable
immunodeficiency syndrome and hypogammaglobulinemia(Table 1).The relative risk of NTM infection varies greatly
depending on the pathology of the host. Thus, Henkle collected
the relative risk values of NTM infection obtained by several
authors, on groups of different patients depending on thepathology of the host, pre-existing NTM infection [3]. These
values of the relative risk of NTM infection and the bibliographic
references used by Henkle can be found in the last two columnsof Table 1.
Table 1. The immunosuppressive conditions and risks for
NTM infections3

HIV/AIDS.

Immunosuppression is a major risk factor for infections
with NTM. During the earlier times of the infection with HIV,disseminated MAC infections appeared in up to 24% of AIDS
patients. Nevertheless with the introduction of highly active
antiretroviral therapy (HAART) in HIV patients, MAC infections
have become less important, while both the increased need fororgan transplants, especially stem cell transplants, as well as
the more frequent use of biologicals and immunosuppressive
therapies in patients with autoimmune diseases have increased
the incidence of NTM infections in these patients [100]. According
2021.31.1 (101 )  Fiziologia - Physiology

to new reports, inhaled steroid therapy increases the risk of
NTM infections in COPD patients and probably less in thosewith bronchial asthma [110]. In contrast to other opportunistic
infections in the context of HIV infection, disseminated MAC
infection only occurs from a CD4+ cell count of less than 50
cells/mm3, which speaks for the importance of the cell-mediatedimmune system in the fight against mycobacteria [109],[110].
M. kansasii can also lead to disseminated NTM infections, but
it is primarily responsible for lung disease in over a half of AIDS
patients [111]. With the introduction of HAART therapy in 1997,
an important reduction in the number of disseminated MAC
infections was achieved. In addition, through the introduction of
antiretroviral therapy, the “immune reconstitution inflammatory
syndrome” has developed, as a result of rebuilding the pathogenspecific immune response during this therapy. In most cases, thissyndrome, which is mainly associated with MAC, presents withfever and painful lymphadenitis [112]. A prophylactic treatment
with 1200mg azithromycin once a week should be currently
given in HIV/patients with CD4+ counts below 50 cells/mm3.
In addition to clarithromycin, ethambutol and/or rifabutin thetreatment for disseminated MAC should also include antivirals tomaintain the underlying immunosuppression under control [113].
A shortened time to death was seen in untreated disseminated
MAC in AIDS patients, but similar survival results were also
found in treated patients had a similar compared to non-MAC
matched controls [3].
Iatrogenic Immunosuppression. It is already known that
glucocorticoids increase the risk of pneumonia in COPD
patients and of tuberculosis by 5-fold [114], while the relative
risks for NTM infections are even higher. In a case-control
study from Oregan, Washington the intake of prednisolone
was 8 times higher among NTM cases compared to controls[109]. Moreover, inhaled glucocorticoids have been linked to asignificantly increased risk of NTM in COPD patients in a study
from Denmark [71], as well as in asthmatic patients in a study
from Japan [115]. In both studies the doses of prednisolone wereabove 15 mg/day and fluticasone above 800 µg/day.
Biologic therapy, that target the TNF signaling pathway,
like the tumor necrosis factor alpha (TNF-alpha)-inhibitors,
are essential in the therapy for autoimmune diseases such as
rheumatoid arthritis, psoriasis and Crohn’s disease. In a study
from the USA, patients receiving a new therapy with infliximab,etanercept or adalimumab had a 5 to 10-fold increased risk of
NTM disease compared to patients with rheumatoid arthritis
without this therapy or to the normal population. The NTM
illnesses were even more common than the tuberculosis inthese patients [114]. Other biological therapies that theoreticallyincrease the NTM risk are rituximab (anti CD20 antibodies),
abatacept (T-cell co-stimulation modulator), tocilizumab (anti-IL-6
antibodies) and ustekinumab (anti-IL-12), but very little safety
data exists on this subject and further studies are required [114].
Unlike tuberculosis, where a benefit of screening for latent
infection before starting an immunosuppresive therapy
have been demonstrated, prospective screening for latent
NTM infections has not been recommended for any of37

immunosuppressed groups [111]. However, CT imaging andrespiratory sampling in searching for pulmonary NTM should beconsidered at least in patients with chronic cough that cannot
be easily explained by usual causes.If possible, discontinuing
immunosuppressive drugs or their substitution with non-biologicaltreatment such as methotrexate or second-line therapies such asabatacept or tocilizumab, which currently present as lower risk
medication, increases the likelihood that antimicrobial therapy for
NTM infections will be successful [112],[109]. A small study fromJapan showed that NTM infections in patients with rheumatoid
arthritis had a positive outcome in those patients who stopped
taking the biological therapy. Furthermore fatal cases wereassociated in this study with underlying lung diseases such as
aspergillosis and interstitial lung disease [3],[113].
Organ transplant. After an organ transplant patients must as
well start an immunosuppressive therapy. The most commonly
administered drugs are mammalian target of rapamycin(mTOR) inhibitor (sirolimus), calcineurin inhibitors (tacrolimus
and cyclosporine), prednisolone and other drugs dependingon the transplanted organ [3]. In a 2014 report that included
293 organ transplants, it was observed that the most NTM
infections occurred after lung transplantation (61%). Heart andliver transplants with similar risks for NTM infections followedwith 26% and 31% respectively and kidney transplants cameon the last place with 17% chances for NTM disease [102]. In
other three studies, MAC and M. abscessus were described as
the most frequently mycobacteria in patients with solid organtransplants with 45% each [105],[115],[116].
NTM infections have also been found in patients withhematopoietic stem cell transplantation (HSCT) in thetreatment of hematological cancers such as leukemia or
multiple myeloma. HSCTs are most frequently associated withcatheter and blood infections, but 18% (17/97) were associatedwith pulmonary NTM in a 2014 review in NTM-infected HSCTpatients. Graft versus host disease was an important risk factor,
present in 46% of NTM infections and corresponding with anincrease in immunosuppressive medications [103]. However,death related to pulmonary and disseminated NTM disease
were in the literature in 7/94 (7.5%) of HSCT patients reported.
Overall, the most frequently NTM in HSCT patients were MAC, M.
abscessus/M. chelonae and M. haemophilum [117],[118],[119].
Therapy in transplanted patients is difficult because of theinteractions between rifampicin, macrolides and aminoglycosides
with calcineurin inhibitors and tacrolimus, and antibiotics should
therefore be chosen very carefully. There have been some
reports of graft vs host reactions in patients who have reduced
immunosuppressive therapy in order to achieve a better immune
system function. This risk should be included in the risk-benefit
assessment in the fight against the mycobacteria [3].

In conclusion, knowing the host risk factors to NTM infection

helps to establish the correct diagnosis and allows theappropriate choice of therapy for lung and disseminated NTM
infection.
38

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Fiziologia - Physiology  2021.31.1 (101)SUSCEPTIBILITATEA GENETICĂ ȘI DOBÂNDITĂ A GAZDEILA INFECȚIILE MICOBACTERIENE NON-TUBERCULOASEREZUMAT
Micobacteriile non-tuberculoase (MNT) sunt microorganisme omniprezente, nepatogene, care produc infecții pulmonare, extrapulmonare și
diseminate doar la persoanele cu deficiențe imune. Dacă infecțiile pulmonare cu NTM apar, în principal, la persoanele imunocompetente
cu boli respiratorii preexistente, infecțiile extrapulmonare și diseminate apar doar la gazdele imunocompromise. Atât factorii de risc locali,
cât și factorii de risc sistemici sunt de o importanță deosebită în patogeneza infecției cu MNT. Sensibilitatea genetică a gazdei joacă unrol important în patogeneza infecțiilor extrapulmonare și diseminate cu MNT, în timp ce susceptibilitatea dobândită a gazdei joacă un rol
important în infecțiile pulmonare, dar și extrapulmonare, precum și diseminate cu MNT. Recunoașterea acestor susceptibilități are un rol
decisiv atât în ​​stabilirea diagnosticului de infecție cu MNT, cât și în aprecierea conduitei terapeutice adecvate.
Cuvinte cheie: MNT, susceptibilitatea genetică și dobândită a gazdei, factorii de risc ai gazdei.

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