Romanian Biotechnological Letters Vol. , No. x, [615242]

Romanian Biotechnological Letters Vol. , No. x,
Copyrigh t © 201 8 University of Bucharest Printed in Romania. All rights reserved
ORIGINAL PAPER

1 Oxidative Burst of Neonatal and Adult Peripheral Blood Phagocytes
Confronting Escherichia coli and Candida albicans

Received for publication, June , 2017
Accepted, June 6, 2017

FLOREDANA -LAURA ȘULAR1,2*#, MIHAELA IANCU3# , MANUELA CUCEREA1,4,
ELENA MOLDO VAN1,4, MARIA LIVIA OGNEAN5, MINODORA DOBREANU1,2
1University of Medicine and Pharmacy of Tîrgu -Mureș, Romania
2Central Laboratory, Emergency Clinical County Hospital of Tîrgu Mureș, Romania
3Department of Medical Informatics and Biostatistics, University of Medicine and Pharmacy
Iuliu Hatieganu Cluj Napoca, Romania
4Regional Center of Neonatal Intensive Care Unit UGON of Tîrgu Mureș, Romania
5Clinical County Emergency Hospital, Sibiu, Romania
*Address for correspondence to: University of Medicine and Phar macy of Tîrgu -Mureș,
Department of Laboratory medicine, Gh.Marinescu street, No.50, Tîrgu -Mureș Romania,
email: [anonimizat]
#Contributed equally to this work.

Abstract
The functional immaturit ies of the cells responsible for the innate defense in neonates have often been
considered a significant risk factor for perinatal infections. The ability of the neutrophils and monocytes to
generate reactive oxygen species (ROS) is mandatory for efficient killing of bacteria and fungi. This study aimed
to assess the production of reactive oxygen species (ROS), as well as the differences in leucocyte populations in
healthy preterm and term neonates, using healthy adults as controls. Burst activation of peri pheral blood
phagocytes (PBPs) was quantified by using a flow cytometry method that implied incubation of peripheral
whole blood phagocytes with various stimuli that included an opsonized Escherichia coli and a non -opsonized
Candida albicans yeast suspensi on. We observed a smaller population of activated granulocytes after exposure
to E. coli in premature neonates than in control adults (41.19±20.63% vs. 58.32±33.46%, p=0.058). Monocyte
burst activation triggered by E. coli, although lower in preterm than i n control adults was not significantly
different (38.97±20.73% vs. 46.82±21.05%, p>0.05). Incubation with non -opsonized C. albicans led to a similar
low granulocyte and monocyte activation in all studied groups, reaching a statistically significant differe nce
only between term neonates and adults. The study shows a deficit of the premature neonate granulocyte to
generate ROS when exposed to opsonized E.coli. The preterm and term, as well as the adult PBPs presented an
almost similar low oxidative burst when exposed to C. albicans, in spite of evoked functional immaturity of the
preterm neonate phagocytes.
Key words: reactive oxygen species, flow cytometry, Escherichia coli , Candida albicans

1. Introduction
The developmental immaturities of the cells respon sible for the innate defense in
neonates have often been considered a significant risk factor for perinatal infections. The
innate immune impairment of the neonate is characterized by a small storage pool of
neutrophils at birth, reduction in serum complem ent activity (A.S. GRUMACH & al. [1]),
low ability to produce antibodies against bacterial antigens and an increased percentage of
“naïve” T lymphocytes. Although monocytes seem to function adequately in neonates, they
have limited chemotactic responses (R .L. SCHELONKA & al. [2]).
Candida species have been described lately as increasingly important pathogens in premature
neonates, especially among very low birth weight infants (VLWB). Invasive Candida

infections are known as the third most common cause of late-onset sepsis in the preterm
immunocompromised populations, its incidence ranging from 7% to 12% (D.K. BENJAMIN
& al. [3] ).
The neutrophil, accompanied by the monocyte, are key effector cells in bacterial and
fungal infections. The ability of the neutr ophils and monocytes to generate ROS is mandatory
for efficient killing of infectious bacteria and fungi (C. FRADIN & al. [4]). Consequently, the
respiratory burst alterations may be a reflection of the neonates’ increased susceptibility to
infections and their detection could be an early diagnostic tool of infection. Some studies
have also shown the decreased ability of the preterm neutrophil to generate an oxidative burst
in response to bacteria (J. KÄLLMAN & al. [5] ).
The opsonization status pays also an important role in granulocyte activation in the
presence of the yeast C. albicans , phagocytosis being quite low in the absence of opsonins
and enhanced greatly when they were introduced in the reaction medium (M. WELLINGTON
& al. [6] ; R.P. GAZENDAM & al. [ 7]). The PBPs of the neonates may have a different
response to Candida than to bacterial stimuli due to the different structure of the fungal wall
and larger size than bacteria.
The objectives of the present study were: i) to identify the impact immaturit y in
preterm and term neonates has on the ability of PBPs to generate ROS in the presence of
various stimuli when compared to healthy adults and ii) to establish how active neonatal
PBPs are in producing ROS when exposed to a bacterial opsonized Escherichi a coli stimulus
in comparison to non -opsonized C. albicans yeast. Opsonins were deliberately omitted from
our study so that the innate function of PBPs could be evaluated at its basic level. We
hypothesized that production of ROS would be more decreased in neonatal PBPs than in
adults when exposed to both E. coli and C. albicans , and that this decrease would be more
obvious in preterm neonates. We considered that this behavior could account for the high
susceptibility of preterm neonates to develop invasive infections.

2. Material and method
Study population and blood samples. The sampled population included 30 preterm
neonates (25 -34 weeks gestational age) and 18 term neonates (37 -41 weeks gestational age)
admitted to the Neonatology Clinic and the Regiona l Center of Neonatal Intensive Care Unit
UGON of the Emergency Clinical County Hospital of Tîrgu Mureș, Romania, as well as a
control group of healthy volunteer adults (n=26). They were selected from the laboratory staff
and chosen by the absence of clinical signs of infection. The studied neonates were recruited
between November 201 4 – September 2015. Only neonates that met the inclusion criteria that
ruled out any sign of inflammation or sepsis during their first 7 days of life were admitted to
the present study (C reactive protein (CRP) levels lower than 10 mg/L and corrected WBC
count >5000/μL and <30000/μL in neonates, I/T (immature nucleated cells/total white blood
cells)  1.16 for the first 24 hours of life and I/T 1.12 within 60 hours of life and a WBC
count <11 000/μL in adults)(G. CHIRICO & al. [8] ; C. CHIESA & al. [9]). Two term and one
preterm neonate were excluded from the study due to difficult sampling that led to
coagulation.
Blood samples for CBC count, lymphocyte count and peripheral blood smears were
collected by heel pricks in K 3EDTA tubes, while blood for burst oxi dation assessment and
CPR quantification was collected by peripheral venipunctures.
The study was conducted according to the World Medical Association Declaration of
Helsinky and was approved by the Ethics Committee of the Emergency Clinical County
Hospita l of Tîrgu Mures, No.19204/29th of September 2014. Informed consent was given by
the mothers of neonates and the volunteer adults.

Blood cell counts and CRP assessment. The complete blood count (CBC) included a
nucleated cell count (Sysmex XT -4000 iTM, Kobe, Japan) platelet count and a peripheral blood
smear 100 -cell differential count. The nucleated cell count represents the total WBC count,
corrected for the presence of nucleated red blood cells. The I/T ratio was calculated for all
neonates as the total percentage of immature neutrophils divided by the total percentage of
neutrophils in the peripheral blood.
The serum CRP levels were assessed by using an automated latex immunoassay
(Architect Plus c4000, Abbott, Chicago, Illinois, USA).
Lymphocyte subs ets quantification. Fifty microliters of well mixed reversed pipetted
whole blood and 20 microliters of a fluorochrome -conjugated monoclonal antibody
(CD3/CD19/CD45, CD3/CD16+CD56/CD45, CD4/CD8/CD3) (Becton Dickinson
Biosciences, San Jose, CA, USA) were we ll mixed in a 5 ml BD round bottom tube and
incubated for 15 minutes on an ice bath in the dark. Two milliliters of lysing solution (FACS
Lysing Solution, BD) was added, followed by gentle vortexing and a 10 minute incubation
period in the dark on a covere d ice bath. Samples were then centrifuged at 300 x g for 5
minutes and then the supernatant was removed. Two milliliters of BD Cell Wash solution
was added and samples were centrifuged at 200 x g for another 5 minutes, followed by
removal of the supernatan t. Upon the remaining cells, 250 μL of 1% paraformaldehyde
solution was added and mixed thoroughly. Samples were stored at 2° – 8°C and analyzed
within 4 hours. Data acquisition and analysis were performed with a FACSCalibur four
colour dual laser flow cyto meter and CellQuest software (BD, San Jose,CA, USA). The
gating strategy was CD45 versus side scatter.
Oxidative burst assay. Whole venous blood specimens collected by venipuncture in
BD sodium heparin tubes were used for quantification of oxidative burst activity of
neutrophils and monocytes. All samples were tested within 1 hour after collection, being kept
meanwhile on a covered ice bath.
The production of ROS was quantified by an in vitro diagnostic flow cytometry
method ( PhagoburstTM, Glycotope Biotech nology ). The kit uses an unlabeled opsonized
Escherichia coli suspension as particulate stimulus (approximatively 1 -2 x 109 bacteria/mL),
the chemotactic peptide N -formyl -methionyl -leucyl -phenylalanine (fMLP) as low
physiological stimulus and the protein k inase C ligand phorbol 12 -myristate 13 -acetate
(PMA) as high stimulus. In order to assess the production of ROS by PBPs that could be
triggered by an invasive fungal infection in the absence of opsonins, we introduced a
supplemetary fungal stimulus for tes ting in the form of a Candida albicans yeast suspension.
Candida albicans ATCC 10321 provided by the Bacteriology Department of our laboratory
was cultured aerobically on Sabouraud chloramphenicol agar for 18 hours at 35°C. An
inoculum of 0.5 optical densi ty C. albicans suspension (approximate cell concentration of 1 -5
x 106 colony forming units/mL) was prepared for each testing round by Using Vitek2
Densichek densit ometer (Biomerieux, France). No opsonins were added to the yeast
suspension. One hundred mic roliters of whole blood, previously kept on an covered ice bath,
no longer than 1 hour after blood collection, were incubated with 20 μl of each of the above
mentioned stimuli for 10 minutes at 37°C. Production of the reactive oxidants during
oxidative bur st was monitored by the addition and oxidation of 20 μl of dihydrorhodamine
123 (DHR123) which served as an oxidative fluorogenic substrate. Burst oxidation was
stopped by adding 2 ml of lysing solution which removed the erythrocytes and led to a partial
fixation of the leucocytes. After centrifugation and one washing step, 200 μL DNA staining
solution was added to exclude aggregation artifacts of bacteria, fungi or cells. The DNA
staining required 10 minutes incubation at 0°C, protected from light. Samples were thus

ready for the FACS analysis that was performed within 30 minutes following DNA staining.
Cells were analyzed by flow cytometry using a 488 nm argon -ion excitation laser. According
to the recommendations of the producer, during data acquisition a “live gate” was set in the
red fluorescence histogram on the events that had at least the same DNA content as a human
diploid cell with the purpose of precluding from analysis bacteria or fungi aggregates that had
the same scatter light properties as the leukocytes. An average number of 15 000 leukocytes
per sample were collected. The percentage of cells that produced ROS (recruitment) was
quantified. The relevant leukocyte cluster was gated in the software program in the scatter
diagram (linear FSC vs lin ear SSC) and its rhodamine 123 green fluorescence was collected
in the FL1 channel (standard FITC filter set) and analyzed. A control sample of a healthy
adult stimulated with saline was used as a negative control to set a marker for fluorescence
(FL1) so that less than 3% of the events were positive. The percentage of activated cells in
the test samples was then set by counting the number of events above this threshold. The final
percentage of activated cells was obtained by subtracting for each sample the negative control
activated cells from the percentage of activated cells stimulated by various stimuli.
Statistical analysis. The percentage of activated cells was considered as a continuous
variable and was expressed by descriptive statistics as mean±sta ndard deviation (SD) or
median and interquartile range [Q1, Q3] where Q1 represented first quartile (25th percentile)
and Q3 -third quartile (75th percentile). The assumption of normal distribution of studied
variables was verified by multiple methods such as Shapiro -Wilk test, Q -Q plot or 95%
confidence interval for univariate skewness and kurtosis estimates.
In order to compare the absolute count of white blood cells (WBC), neutrophils,
monocytes and lymphocytes and the percentage of lymphocyte subclasses (total T, T helper,
T cytotoxic, B and NK lymphocytes) between studied groups, we used the ANOVA method
or Welch test considered as an appropriate approach for heterogeneity of variance in our data.
Because we were interested in comparing the distribution of the percentage of activated cells
triggered by different intra group stimuli (adults), the Student -t test for dependent samples
was performed.
In order to compare the percentage of activated cells caused by different stimuli inter
groups (premature neo nates vs. adults and premature vs. term neonates), we used the
parametric Student -t test for independent samples or the nonparametric Mann -Whitney test.
Because of multiple comparisons released, we reported adjusted p -values for
appropriate statistical te sts in order to maintain the family error rate to the alpha value of
0.05.
For all two -sided statistical tests for group comparisons, the statistical significance
was achieved if adjusted p -values≤ 0.05.
The statistical analysis was performed with the advanced software environment for
statistical computing and graphics, R version 3.2.4 (R Foundation for Statistica l Computing,
Vienna, Austria), STATISTICA (StatSoft, USA, version 6) and MedCalc version 17.

3. Results
Clinical characteristics of the studied neonatal samples (see Table 1). The studied
preterm and term neonatal samples presented no differences regardin g gender and maternal
age (p>0.05). Birth weight was significantly lower in preterm neonates (p<0.0001) and
preeclampsia was a condition that affected only 13.33% of the mothers of the studied preterm
neonates. The caesarean way of delivery was more often encountered in the case of preterm
neonates, more than half of them (60%) being delivered this way compared to term neonates
(20%) (p=0.0167). Tocolytic medication was administered to two mothers of the preterm and
one of the term neonates, while dexametha sone prophylaxis was used for half of the mothers

of the preterm neonates. Chorioamnionitis and systemic infection were not present in any of
the mothers of either neonatal group. Prophylactic antibiotic therapy was administered for
ruptured membranes to 3 0% of the mothers of the preterm neonates.

Table 1. Clinical characteristics of the studied neonates
Preterm Term p
Number n=30 n=18
Maternal age
(mean±SD, years) 26.21±1.36 26.88±1.8 0.7677*
Delivery
(Vaginal/Caesarean )(%) 12/18 14/4 0.0167**
Male/female gender (%) 10/16 11/7 0.2199
Gestational age
(mean±SD, weeks) 30.15±2.7 38.85±1.14 P<0.0001 
Birth weight
(mean±SD , grams) 1388±443.5 3239±440.9 P<0.0001 
Maternal conditions
Preeclampsia – n (%) 4 (13.33%) 0 ND
Chorioamnionitis – n (%) 0 0 ND
Systemi c infection – n (%) 0 0 ND
Diabetes – n (%) 0 0 ND
Maternal medications
Corticoid prophilaxis (DXM) – n (%) 15 (50%) 0 ND
Tocolitics – n (%) 2 (7.69%) 1 (5.55%) 1.000
Antibiotics – n (%) 9 (30%) 0 ND
Student -t test for independent samples; Fisher’s Exact test; SD=standard deviation; ND=not determined;
DXM=dexamethasone

WBC and lymphocyte subclasses in neonates and adults. We identified a significant
difference between the absolute count of WBC in preterm and term neonates when compared
to ad ults (p=0.029, and p=0.003 respectively), the mean of corrected absolute count of WBC
value being greater in the term neonate group than in preterm neonates and adults (Table 2).
We found also a significant difference between the mean of neutrophil absolut e count in term
neonates and adults (p=0.015), but there was no difference identified between preterm
neonates and adults. The mean of neutrophil count was greater in the term neonate group than
in adults (Table 2).
Table 2. WBC and lymphocyte subclasses in studied groups
Preterm neonates Term neonates Adults
WBC (x 103/μL) 8.88±4.65 13.68±7.59 6.57±1.76
Neutrophils (x 103/μL) 4.13±3.04 7.89±5.49 3.76±1.27
Monocytes (x 103/μL) 1.32 [0.84, 1.63] 1.88 [1.06, 2.31] 1.00 [0.00, 1.00]
Lymphocytes (x 103/μL) 3.32±1.37 3.98±1.42 2.03±0.61
B (%) 13.53±8.63 10.73±4.73 10.33±3. 49

NK (%) 6.923±3.93 10.18±9.86 14.26±7.86
T (%)
Th (%T) 77.55±9.07 77.69±12.8 73.39±5.69
73.88±9.01 74.34±5.67 58.35±9.93
Tc (%T) 23.29±9.30 22.41±5.17 33.65±8.22
The absolute lymphocyte counts were significantly different in preterm and neonates
when compared to adults (p<0.001), the estimated mean value of the lymphocyte absolute
count being greater in term neonates (Table 2). In both preterm and term neonatal
populations, the absolute monocyte count was significantly higher than in healthy adults
(p<0.001).When comparing the frequencies of B, T and NK lymphocyte subclasses (Tc), no
differences were detected between the studied preterm, term neonates and adults except for a
higher frequency of the NK lymphocyte subclass in preterm neonates when comp ared to
adults (p=0.001).
PBPs burst in adults. To establish the normative adult pattern of oxidative burst
induced by opsonized E. coli and non -opsonized C. albicans , 26 healthy adults were used as
controls during the study period. Data were corrected to the background activity of the same
PBPs preparation incubated in the absence of any stimulus with saline. The adult neutrophils
exposed to E. coli exhibited an approximately 60% increase in burst activity relative to
control neutrophils (p=0.004). The no n-opsonized C. albicans yeast did not influence
significantly the oxidative burst activity of adult neutrophils when compared to the absence
of stimulus (p>0.05) (data not shown).
Monocyte burst activation in adults in the presence of opsonized E. coli was on
average 44% higher than saline control activation (p=0.004), while the monocyte burst
activation in adults in the presence of non -opsonized C. albicans was on average 1.98%
greater than saline control activation but without statistical significance (p =0.128).
PBPs burst in neonates versus adults. We found a significant difference in the
distribution of the percentages of spontaneously activated granulocytes between term and
preterm neonates compared to adults (p<0.05). We also noticed that median (inte rquartile
range: [Q1, Q3]) percentages of spontaneously activated granulocytes were 9.44 [2.63, 20.88]
for term neonates and 5.61 [2.65, 14.84] for preterm neonates versus 2.32 [1.84, 2.51] in
adults. We observed greater point estimations of the median in preterm and term neonates
versus adults groups (Table 3).

Table 3. Burst activation of granulocytes and monocytes triggered by exposure to various stimuli
Preterm neonates Term neonates Adults
Activated
granulocytes
(%) Activated
monocytes (%) Activa ted
granulocytes
(%) Activated
monocytes
(%) Activated
granulocytes
(%) Activated
monocytes
(%)
Saline
solution 5.61
(2.65 -14.84 ) 1.57
(0.74 -2.86) 9.44
(2.63 -20.88 ) 1.79
(0.31 -2.84) 2.32
(1.84 -2.51) 1.12
(0.41 -1.68)
fMLP 5.51
(1.27 -16.70 ) 2.70
(1.31 -5.72) 6.05
(2.24 -19.65 ) 1.61
(1.04 -4.82) 1.46
(0.56 -4.42 0.41
(-0.06-1.57)
PMA 86.33
(75.63 -93.46 ) 64.77
(36.22 -91.65) 81.62
(72.76 -94.13 ) 66.89
(22.76 -95.15) 91.49
(81.42 -96.90 ) 49.25
(29.37 -76.30)
E. coli 41.19±20.63 38.97±20.73 50.01±26.50 39.85±24.36 58.32±33.46 46.82±21.05
C. albicans 3.02
(1.87 -4.53) 2.34
(1.11 -3.27) 4.35
(1.69 -12.69 ) 2.11
(0.51 -6.45) 2.03
(1.31 -3.51) 1.44
(0.62 -3.76)

We identified a difference with a tendency towards statistical significance between
the granulocyte activation triggered by E. coli in premature neonates compared to the control
adults (41.19±20.63% vs. 58.32±33.46%, p=0.058) . Though the granulocyte activation
degree was lower in premature neonates when stimulated with the E. coli suspension than in
term neonates, in our study it did not reach a statistically significant difference
(41.19±20.63% vs. 50.01±26.50%, p=0.472).
We found no difference regarding the behavior of the preterm and term granulocytes
in the presence of the physiological fM LP stimulus when compared to the adult granulocytes.
We did notice a higher degree of PMN activation in the presence of the physiological fMLP
stimulus in preterm and term neonates than in the adult granulocytes (5.51 [1.27, 16.70]
preterm vs. 6.05 [2.24, 19.65] term vs. 1.46 [0.56, 4.42] adults), though not statistically
significant (p>0.05).
There was no difference concerning PMN activation by the chemotactic peptide PMA
in preterm and term neonates compared to adult granulocytes (adjusted p>0.05). When t he
chemotactic peptide PMA was used as a stimulus, granulocytes in all the studied groups
reached a similar maximal degree of activation: 86.33 [75.63, 93.46] preterm vs. 81.62
[72.76, 94.13] term vs. 91.49 [81.42, 96.90] adults.
When examining the effect non-opsonized C. albicans yeast incubation had on
granulocyte activation, we identified no difference in preterm and term samples when
compared to adults (p>0.05). The unopsonized C. albicans stimulus caused a low granulocyte
activation in all three studie d groups regardless of their degree of maturation (3.02 [1.87,
4.53] in preterm vs. 4.35 [1.69, 12.69] in term vs. 2.03 [1.31, 3.51] in adults). Although the
low granulocyte activation degree was similar, it reached a statistically significant difference
between the term neonates and adult study group ( p=0.034) (see Figure 1).

050100
neutrophils neutrophils monocytes monocytes
Escherichia coli Candida albicans Preterm neonates
Term neonates
Adults% of activated phagocytes

Figure 1. Neutrophil and monocyte activation triggered by E. coli and C. albicans . The box –
plots contain medians and minimum -maximum values of activated PBPs

Examinin g the spontaneous activation of the monocyte population in the presence of
the saline solution, we detected no statistical significance among the premature neonates vs.

term and premature vs. adults (adjusted p>0.05). The median [Q1, Q3] percentages were 1 .57
[0.74, 2.86] in preterm and 1.79 [0.31, 2.84] term vs. 1.12 [0.41, 1.68] in adults.
There was a mean difference with a tendency towards statistical significance between
the percentage of monocytes activated by exposure to E. coli in premature neonates compared
to the control adults (38.97±20.73% vs. 46.82±21.05%, p>0.05) . Although the monocyte
activation degree was lower in premature neonates when stimulated with the opsonized E.
coli than in term neonates, in our study it did not reach a statisticall y significant difference
(38.97±20.73% vs. 39.85±24.36%, p>0.05).

4. Discussions
Many facets of neonatal immune deficiency contribute to sepsis and other infectious
syndromes in the newborn. Because of the important role the production of ROS by PBPs has
in defense, we chose to focus on this defensive mechanism.
In our study, although the preterm neonates showed a lower capacity to up -regulate
the ability of the neutrophil to generate ROS than term neonates when exposed to the E. coli
stimulus, the differ ence in neutrophil recruitment had a tendency towards statistical
signification only when compared with healthy adults. We believe that this dif ference in
activation could become significant if we could have accessed larger study groups. Although
the unlab eled E. coli stimulus provided by the commercial kit was opsonized in order to
facilitate rapid granulocyte activation, our results show that the percentage of oxidative active
neutrophils in preterm neonates remains low even when using whole blood which c ontains
neonatal humoral factors such as complement and immunoglobulins.
Our results are consistent with the ones described by similar studies that focused on
other living microbes such as coagulase -negative Staphylococci and Streptococcus
epidermidis. BJÖRKQVIST & al. described a lower percentage of oxidative active
neutrophils after bacterial stimulation in preterm compared with term newborns, but the
difference they found was not statistically significant. When they compared the mean
fluorescent intensit y in the preterm and term neonates , it showed as highly statistically
significant in favor of term neonates for all studied bacterial strains (M. BJÖRKQVIST & al.
[10]).
Candida albicans infections are life -threatening conditions and a common cause of
late-onset sepsis in premature neonates. They are associated with significant morbidity,
morality, health related expense and neurodevelopmental impairment (K.G. DESTIN & al.
[11]). According to the published data, invasive Candida infections affect immunosup pressed
patients and those with defects of the immune system.
Previous studies performed on preterm and term neonates have focused mainly on the
quantification of oxidative burst activity of PMN by using bacteria as particulate stimulus.
Because bloodstrea m infections due to Candida species are a major cause of morbidity and
mortality in neonates and children, nosocomial candidemias ranging from 10% -20%, we have
decided to study one of the first steps of the non -specific cellular immune response that
consis ts in PBBs activation and production of ROS when stimulated with living Candida
albicans yeast cells. Our results have shown a very low activation of PBPs and consecutive
low production of ROS both in adults and neonates when live C. albicans yeast cells w ere
used as particulate stimulus. In some cases, burst activation in the presence of living C.
albicans was even lower than the spontaneous activation of PBPs verified by the saline
solution tube, thus indicating an inhibitory effect of the yeast cells upo n the production of
ROS. In our study, this low activation of PMN and monocyte s was similar in all three studied
groups with no detected statistically difference.

In our data set, the analysis of the lymphocyte subclasses has revealed a slightly
higher bu t not significant difference in the frequency of CD19+B lymphocytes as well as a
significantly higher frequency of CD4+T lymphocytes (Th) in healthy preterm and term
neonates when compared to adults. Our results are concordant with data provided by previou s
studies on the phenotypic differences in leucocyte populations among healthy preterm and
full-term neonates (C. QUINELLO & al. [12]). We identified a significantly lower
percentage of CD8+T lymphocytes (Tc) in both preterm and term neonates than in adult s (see
Table 2).
Studies have shown that there are additional variables that could impact ROS
production. The study conducted by WELLINGTON & al. showed that live C. albicans
suppresses primary phagocyte production of ROS despite of the proven stimulatory effects of
the fungal surface structures (1,3 --glucan and 1,4 --glucan) upon ROS production through
dectin -1 activation (I. RUBIN -BEJERANO & al. [13] ; P. BONFIM -MENDONÇA & al.
[14]). The study also showed that live C. albicans scavenged more ROS than hea t killed C.
albicans . Nevertheless, due to the fact that live and heat killed Sacharomyces cerevisiae
scavenging was similar to that observed with C. albicans , they concluded that scavenging
alone cannot account for the observed suppression of ROS producti on (M. WELLINGTON
& al. [15]).
Other studies, such as the one published by FROHNER & al. have concluded that
ROS induction is independent of morphology as both yeast and hyphal forms of C. albicans
trigger ROS in primary innate immune cells such as macroph ages and dendritic cells ( I.E.
FROHNER & al. [16]). Nevertheless, studies have shown that C. albicans cell surface
superoxide dismutase encoded by the Sod5 gene degrades extracellular ROS produced by
immune cells, thus overriding the glucan burst activati on (I.E. FROHNER & al. [16]).
Neutrophils seem to induce also Sod5 expression, although they inhibit the yeast -to-hyphal
formation in C. albicans (P. MIRAMÓN & al. [17] . These assertions are consistent with our
research that describes a low ROS production and even burst inhibition in some cases in
PMN and monocytes when C. albicans was used as particulate stimulus .
Our results are also consistent with other studies such as the one of YOST & al . which
has shown the importance of NETs (neutrophil extracellul ar traps) formation by PMN in
order to induce an adequate non -specific cellular immune response. These DNA complexes
which are thought to form via a unique death pathway signaled by nicotine adenine
dinucleotide phosphate (NADPH) oxidase -generated ROS, acc omplish both capture and
killing of bacteria and fungi, which are antimicrobial activities that can be suppressed by
endonucleases and DNases expressed by some organisms. The study has also revealed that
term neonatal PMN failed to produce NETs when incuba ted with live E. coli , Staphylococcus
aureus and PMA in contrast to adult neutrophils. This phenomenon was associated with
impaired bacterial killing and thus it could contribute to the pathogenesis of severe infections
in early life ( YOST & al. [18] ). Als o, generation of ROS did not complement the defect in
NET formation by neonatal neutrophils as it happened in adult cells with inactivated NADPH
oxidase, proving that ROS are necessary, but not sufficient signaling intermediaries. Thus,
the defective ROS p roduction that we have identified in the studied neonatal groups seems to
be accompanied also by insufficient bacterial killing due to a low NETs production in
neonates. Also, others studies have shown that while the killing of opsonized C. albicans is
dependent upon production of ROS by the NADPS oxidase system, unopsonized C. albicans ,
as in our case, is dependent solely on the complement receptor 3 (CR3) and the signaling
proteins phosphatidylinositol -3-kinase and caspase recruitment domanin -containing protein 9
(CARD9)(R.P. GAZENDAM & al. [7] ).

Commercial methods such as the one used in our study (PhagoburstTM, Glicotope) allow
exact quantification of granulocyte and monocyte ROS production by fluorometric analysis
using whole blood, thus making the met hod more approachable. The assay that uses
dihydrorhodamine 123 as a flourogenic substrate has already been validated and its efficiency
in correctly distinguishing patients suffering from granulocyte defects has been verified by
previous studies (A. LUNN & al. [19]), providing results within a short period of time,
usually within a few hours. Considering the severity and the rising frequency of fungal
systemic infections among neonates, inspite of adequate diagnosis and treatment, we have
considered comple ting the commercial kit we used with C. albicans as an extra fungal
stimulus. Our results may not be very relevant because of the complete lack of opsonins used
in the study, WELLINGTON & al. having clearly proven that phagocytosis, and by this we
assume t hat bust oxidation also, is clearly enhanced in the presence of these molecules
(WELLINGTON & al. [6]).
Limitations of the study. The limitation of our study consists in the relatively small
samples of patients that we could assess, as well as the rather v ariable number of cells that
could be analyzed per subject due to the variable number of WBC of the recruited neonates.

5. Conclusions
The present study showed a deficit in ROS production of the premature neonate
granulocyte when exposed to opsonized E. coli. A similar behavior could not be identified in
preterm or term PBPs compared to adult PBPs when non -opsonized C. albicans was used as a
stimulus. The immaturity of the preterm granulocytes and monocytes did not distinguish
greatly their almost absent response to fungal stimuli from the low activation of adult PBPs .
6. Acknowlegements
This study was supported by the Research Grants of the University of Medicine and
Pharmacy of Tîrgu Mureș, Romania, Project number 15/23.12.2014. The authors thank the
medical doctors and nurses of the Regional Center of Neonatal Intensive Care Unit UGON
and Neonatology Department of Tîrgu Mureș for the help provided with the selection of
adequate study candidates and blood collection. We thank also An ca Bac ârea, M.D., for the
evaluation of blood smears and the laboratory technicians that helped with the experiments.

Disclosure
The authors declare no conflicts of interest.

References
1. GRUMACH AS, CECCON ME, RUTZ R, FERTIG A, KIRSCHFINK M. Complement profile in
neonates of different gestational ages. Scand J Immunol 2014,79(4):276 –81.
2. SCHELONKA RL, INFANTE AJ. Neonatal immunology. Semin Perinatol 1998 Feb,22(1):2 –14.
3. BENJAMIN DK, STOLL BJ, FANAROFF AA, MCDONALD SA, OH W, HIGGINS RD, et al.
Neonatal candidiasis among extremely low birth weight infants: risk factors, mortality rates, and
neurodevelopmental outcomes at 18 to 22 months . Pediatrics 2006 Jan,117(1):84 –92.
4. FRADIN C, DE GROOT P, MACCALLUM D, SCHALLER M, KLIS F, ODDS FC, et al.
Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in
human blood. Mol Microbiol 2005 Feb 18,56(2):397 –415.
5. KÄLLMAN J, SCHOLLIN J, SCHALÈN C, ERLANDSSON A, KIHLSTRÖM E . Impaired
phagocytosis and opsonisati on towards group B streptococci in preterm neonates . Arch Dis Child Fetal
Neonatal Ed 1998 Jan,78(1):F46 -50.
6. WELLINGTON M, BLISS JM, HAIDARIS CG. Enhanced phagocytosis of Candida species mediated
by opsonization with a recombinant human antibody singl e-chain variable fragment . Infect Immun
2003 Dec,71(12):7228 –31.

7. GAZENDAM RP, HAMME JL VAN, TOOL ATJ, HOUDT M VAN, VERKUIJLEN PJJH, HERBST
M, ET AL. Two independent killing mechanisms of Candida albican s by human neutrophils : evidence
from innate immunity defects. Blood 2014,124(4):590 –8.
8. CHIRICO G, LODA C. Laboratory aid to the diagnosis and therapy of infection in the neonate.
Pediatric reports 2011, 3(Table 1):1 –5.
9. CHIESA C, PANERO A, OSBO RN JF, SIMONETTI AF, PACIFICO L, OSBORN JF, et al.
Diagnosis of neonatal sepsis: a clinical and laboratory challenge . Clin Chem 2004 Feb 1,50(2):279 –87.
10. BJÖRKQVIST M, JURSTRAND M, BODIN L, FREDLUND H, SCHOLLIN J . Defective Neutrophil
Oxidative Burst in Preterm Newborns on Exposure to Coagulase -Negative Staphylococci. Pediatr Res
2004 Jun,55(6):966 –71.
11. DESTIN KG, LINDEN JR, LAFORCE -NESBITT SS, BLISS JM. Oxidative burst and phagocytosis of
neonatal neutrophils confronting Candida albicans and Cand ida parapsilosis . Early Hum Dev 2009
Aug,85(8):531 –5.
12. QUINELLO C, SILVEIRA -LESSA AL, CECCON MEJR, CIANCIARULLO MA, CARNEIRO –
SAMPAIO M, PALMEIRA P. Phenotypic differences in leucocyte populations among healthy preterm
and full -term newborns. Scand J I mmunol 2014,80(1):57 –70.
13. RUBIN -BEJERANO I, ABEIJON C, MAGNELLI P, GRISAFI P, FINK GR. Phagocytosis by human
neutrophils is stimulated by a unique fungal cell wall component. Cell Host Microbe 2007 Jul
12,2(1):55 –67.
14. BONFIM -MENDONÇA P DE S, RATT I BA, GODOY J DA SR, NEGRI M, LIMA NCA DE,
FIORINI A, ET AL. β -Glucan induces reactive oxygen species production in human neutrophils to
improve the killing of Candida albicans and Candida glabrata isolates from vulvovaginal candidiasis.
PLoS One 2014,9(9) :e107805.
15. WELLINGTON M, DOLAN K, KRYSAN DJ. Live Candida albicans suppresses production of
reactive oxygen species in phagocytes. Infect Immun 2009 Jan,77(1):405 –13.
16. FROHNER IE, BOURGEOIS C, YATSYK K, MAJER O, KUCHLER K . Candida albicans cell
surface superoxide dismutases degrade host -derived reactive oxygen species to escape innate immune
surveillance . Mol Microbiol 2009,71(1):240 –52.
17. MIRAMÓN P, KASPER L, HUBE B. Thriving within the host: Candida spp. interactions with
phagocytic cells. Med Microbiol Immunol 2013 Jun 25,202(3):183 –95.
18. YOST CC, CODY MJ, HARRIS ES, THORNTON NL, MCINTURFF AM, MARTINEZ ML, et al.
Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human
neonates. Blood 2009 Jun 18, 113(25):6419 –27.
19. LUN A, SCHMITT M, RENZ H . Phagocytosis and oxidative burst: Reference values for flow
cytometric assays independent of age. Clin Chem 2000,46(11):1836 –9.