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IMPAIRED PHAGOCYTOSIS AND OXIDATIVE BURST ACTIVITY OF
MONOCYTES ON ESCHERICHIA COLI INFECTION IN THALASSEMIA
Chayada Thiengtavor,1 Sirikwan Siriworadetkun,
2 Kittiphong Paiboonsukwong,
2
Suthat Fucharoen,2 Kovit Pattanapanyasat,
3 Saovaros Svasti,
2 Pornthip Chaichompoo,
1,*
1Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand;
2Thalassemia Research Center, Institute of Molecular biosciences, Mahidol University,
Nakhon Pathom, Thailand 3Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok, Thailand.
*e-mail: pornthip.chh@mahidol.ac.th
___________________________________________________________________________
Abstract:
Thalassemia is a common genetic disorder worldwide, with at least 60,000 severely affected
individuals born every year. Infection is the first cause of death (46%) in developing country
and the second cause of death (12.6%) in developed country, which Escherichia coli as major
cause of severe infection (26%) and Klebsiella pneumoniae as minor (23%).
Monocytes/macrophages are the main initial effectors of the innate immunity that can clear
the invading pathogen and activate adaptive immunity through phagocytosis. Herein,
thalassemic monocyte function on clearance ex vivo E. coli infection was investigated. A
method for duo-measurement of phagocytosis and oxidative burst in whole blood using three-
color flow cytometric method was developed. Lipopolysaccharide-primed whole blood
samples showed increased phagocytosis and oxidative burst activity of phagocytes as
measurement of percentages and mean fluorescent intensity of propidium iodide labeled E.
coli and 2′,7′-dichlorofluorescin diacetate when compared to unprimed as dose- and time-
dependent manner. Thalassemic monocytes had significant decreased efficiency on
phagocytosis (13.9±5.2%) and oxidative burst (1.2±0.6%) when compared to normal subjects
(44.3±10.4 and 12.8±6.8%, respectively, P < 0.05). Decreased phagocytosis could affect to
pro-inflammatory and immunogenic peptide presentation to activate humoral and cell-
mediated immune responses. These findings suggest that impaired phagocyte function of
monocytes in thalassemia supports the clinical observation as severe anemia with
splenectomized patients has an increased risk of infection. This study may provide a basic for
monocyte/macrophage-centered therapeutic strategies in thalassemia. __________________________________________________________________________________________
Keywords : innate immunity, phagocytosis, oxidative burst, bacterial infection, flow
cytometry
Introduction :
Thalassemia is a common genetic disorder worldwide, with at least 60,000 severely affected
individuals born every year. In Thailand, approximately 3,000 children are born each year
with the disease and 100,000 cases are reported in the population (Fucharoen and
Winichagoon, 1987). Patients are suffering from severe anemia and other complications such
as growth retardation, severe bone changes, hepatosplenomegaly, heavy iron overload,
osteoporosis, endocrinopathies, heart failure, pulmonary hypertension, thromboembolic
events, immune abnormalities and infection. Infection is a major complication and the
leading cause of death in thalassemia, which Escherichia coli (26%) and Klebsiella
pneumoniae (23%) as major pathogens. General management for prevention of infection in
thalassemia is treatment with the immunization with pneumococcal and hepatitis vaccines,
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oral penicillins especially in patients with splenectomy, removal of predisposing factors such
as gallstones, iron overload and appropriate antibiotics. Albeit largely improvement of
prognosis and therapeutics to protect infectious diseases, infection are still claim the lives of
too many thalassemic patients. Several factors are associated with immune abnormalities of
both innate and adaptive immunity on β-thalassemia patients such as severe anemia, multiple
blood transfusions, iron overload, iron chelator treatment and splenectomy. Desferrioxamine
(DFO) as iron chelator therapy might have increased risk for Yersinia enterocolitica infection
which may be localized to mesenteric nodes and tonsils or occur as a generalized form such
as septicemia (Wanachiwanawin, 2000). Defect in absolute number and function of immune
cells such as increased amounts and activity of CD8 suppressor T cells, decreased CD4/CD8
ratio, reduced T cell proliferation, increased amounts and activity of B cells, defective
chemotaxis and phagocytosis of neutrophils and macrophage and defective natural killer cell
function in β-thalassemia patients with susceptibility to infection have been reported
(Pattanapanyasat et al., 2000; Vento et al., 2006). Unfortunately, the mechanism of immune
abnormalities is poorly understood. It is important to notice that abnormal red blood cell
(RBC) membrane in thalassemia is continuously cleared by monocytes and macrophages that
eventually lead to hyperphagocytosis and hypersplenism. The standard approach for treating
hypersplenism is splenectomy. Unfortunately, it is increased risk to infection in thalassemia.
Additionally, commonly observed in long-term receipt of blood transfusions leads to
continuous alloantigenic stimulation that is associated with autoimmune hemolysis, T cell
and B cell changes and modification of monocyte and macrophage function (Vento et al.,
2006). Finally, defective functions of neutrophils, natural killer cells and complement system
have also been reported (Wanachiwanawin, 2000).
Monocytes/macrophages are cells in the innate immunity and play an important role of
antigen-presenting cells and release many inflammatory cytokines and chemokines that
contribute to infiltrate immune cells into site of infection and initiate the adaptive immunity.
Lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative
bacteria, is a common antigen to activate immunity. During infection, patients with sepsis
due to gram-negative bacteria infection had 0.5-5 ng/mL plasma LPS (Bohmer et al., 1992).
Human monocytic cell line, THP-1, treated with LPS at low dosages (< 1 ng/mL) of leads to
increased synthesized the pro-inflammatory mediator interleukin (IL)-6, while higher dosages
(> 10 ng/mL) induced the expression of both IL-6 and IL-33 (Morris et al., 2014).
Monocytes/macrophages recognize and bind bacteria via microbe-associated molecular
patterns (MAMPs) and initiate phagocytosis. The phagocytosis is initiated by opsonizing
microbes with antibodies that bind with high-affinity to Fc receptor (CD16) on monocytes/
macrophages. Complement components are another predominant factor in blood circulation
that enables efficient opsonization. The C3b complement binds to surface of microbe act as
opsonin and recognized by macrophage-1 antigen (Mac-1). Mac-1 is a heterodimeric integrin
of two subunits; alpha-M (CD11b) and beta-2 (CD18). When the interaction between
microbe and receptor on monocytes/macrophages occur, the plasma membrane of monocytes/
macrophages begins to redistribute and form the cup shaped projection around microbe and
pinch off the interior of the cup to from an inside-out intracellular vesicle. Upon phagosome
closure, the maturing phagosome traverses an early and late phagosomal and a
phagolysosomal stage paralleling endosomal maturation. In the lumen of phagosome is
acidification by the proton pumping vesicular ATPase (Weiss and Schaible, 2015). The
phagocytosed microbes are destroyed to generate the peptides and presented to T cell via
major histocompatibility complex (MHC) to initiate adaptive immune response.
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In this study, the duo-measurement of phagocytosis and oxidative burst of monocytes
followed E. coli treatment in peripheral blood samples using three-color flow cytometric
method was developed. This procedure is based on an assay described earlier (Bohmer et al.,
1992; Keawvichit et al., 2012), which exploits the use propidium iodide (PI) for staining
bacteria, a fluorescent dye, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), as reactive oxygen
species (ROS) marker and monoclonal antibody specific to glycoprorin A (GPA) for
excluded immature RBCs, intact RBCs or ghost cells.
Methodology :
Subjects
Forty β-thalassemia/HbE patients (12 splenectomy and 16 non-splenectomy) and 12 normal
subjects with the age ranged from 24 to 48 years were recruited. This study approved by the
Mahidol University Central Institutional Review Board (MU-CIRB), approval number
2015/052.0104. Written informed consent was obtained from all individual participants
included in the study. All subjects had no hydroxyurea, prednisolone, blood transfusion and
iron chelator before blood sample collection at least 4 weeks. All blood samples were
collected at room temperature (RT) and processed within 2-3 h. Hematological parameters
and differential white blood cell (WBC) count were described in Table 1.
Table 1 Hematological parameters
Description Normal subjects -Thalassemia/HbE patients
Non-splenectomy Splenectomy
Number 12 16 12
RBC count (×106/L) 4.5 ± 0.2 4 ± 1.0* 3.3 ± 0.6*
Hb (g/dL) 13.3 ± 1.0 7.3 ± 1.5* 6.5 ± 1.2*
Hct (%) 39.7 ± 2.6 23.7 ± 4.5* 23.1 ± 3.2*
MCV (fL) 86.1 ± 4.5 59.7 ± 7.0* 70.3 ± 6.7*
MCH (pg) 28.8 ± 1.8 18.3 ± 2.5* 19.9 ± 2.3*
MCHC (g/dL) 33.5 ± 1.0 29.5 ± 5.4* 28.3 ± 1.6*
% RDW 12.5 ± 0.6 25 ± 3.5* 24.9 ± 2.1*
WBC count (×103/L) 6.5 ± 1.9 5.4 ± 2.1 10.4 ± 6.3
WBC differential count
%Blast cells 0 ± 0.4 0 ± 0.6 1 ± 1.3
%Neutrophils 62 ± 5.7 59 ± 12.3 48 ± 12.7*
%Eosinophils 2 ± 0 2 ± 2.2 2 ± 1.5
%Basophils 0 ± 0 0 ± 0 0 ± 0.3
%Lymphocytes 27 ± 7.3 29 ± 15.4 40 ± 14.9*
%Monocytes 8 ± 5.8 9 ± 6.8 10 ± 7.1
NRBCs (/100WBC) 0 ± 0 43 ± 66.3 456 ± 204.7*
Platelet count (×103/L) 270 ± 49.1 182 ± 96.7* 598 ± 142.9*
%Reticulocyte count 0.9 ± 0.2 6.7 ± 3.8* 13.9 ± 5.5*
Reticulocytest (×109/L) 39.8 ± 9.1 254.3 ± 135.4* 441 ± 136.6*
%HbA2/HbE 2.8 ± 0.3 54.6 ± 12.6* 53.5 ± 10.1*
%HbF 1.1 ± 0.8 32.9 ± 15* 34.7 ± 11*
%HbA 86.7 ± 0.8 21.5 ± 10.5* 19.6 ± 1.3*
RBC = red blood cells, Hb = hemoglobin, Hct = hematocrits, MCV = mean corpuscular volume, MCH = mean
corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, WBC = white blood cells,
NRBCs = nucleated red blood cells. *Significant different at P < 0.05 when compared to normal subjects
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Propidium iodide staining bacteria
Escherichia coli stain DH5α was fixed with cold-70% ethanol solution for 2 h at 4°C. After
incubation and wash, fixed E. coli was kept as master stock at 2×109 bacteria/mL in
phosphate buffer saline (PBS, Hyclone). One milliliter of fixed bacteria was stained with
100 μg/mL PI (Sigma) for 30 min at 4°C and used within 1 h.
Treatment of monocytes with Escherichia coli
Four hundreds forty microliter of whole blood samples in CPDA-1 anticoagulant were
primed with 1 and 10 ng/mL LPS (Sigma) for 60 min at 37°C. Then samples were pre-
incubated with 50 μM DCFH-DA (Sigma) for 10 min at 37°C. Fifty microliter of samples
were collected for analysis of dye loading and endogenous ROS levels (DCFT10), and then,
remained samples were co-incubated with PI stained E. coli (at 0.1-10×107 bacteria in 500 μl
final volume) for 0-180 min at 37°C (T0 to T180).
Phagocytosis and oxidative burst analysis
Each sample was placed in 1 mL 1×FACS lysing solution (BD Biosciences) for 10 min at
RT, after centrifugation, pellet samples were stained with PE conjugated anti-GPA (BD
Biosciences) by followed manufacture’s recommended. Data from at least 50,000 events of
leukocytes were acquired by BD FACScan flow cytometer (BD Biosciences) and analyzed
by BD CellQuest software (BD Biosciences) within 1 h after staining. The first analyzed
using the log amplification of FL-2 channel for gating negative population of GPA
(leukocytes) (Figure 1A). Then, FSC-H/SSC-H analysis of size and granularity was separated
leukocytes into granulocytes, monocytes (R2 region) and lymphocytes (Figure 1B). The
percentages and mean fluorescent intensity (MFI) of phagocytosis and oxidative burst were
analysis by quadrant analysis of FL-3 and FL-1 channel (Figure 1C-I).
CD16 and CD11b expression analysis
Fifty microliter of whole blood samples were placed in 1 mL 1×FACS lysing solution (BD
Biosciences) for 10 min at RT, after centrifugation, pellet samples were stained with PE
conjugated anti-GPA (BD Biosciences), BV421 conjugated anti-CD16 (BioLegend), BV510
conjugated anti-CD11b (BioLegend) and BV605 conjugated anti-CD45 (BioLegend) by
followed manufacture’s recommended. Data from at least 50,000 events of leukocytes were
acquired by BD FACS LSRII flow cytometer (BD Biosciences) and analyzed by BD Diva
software (BD Biosciences). The first analyzed using the log amplification of FL-2 channel
for gating negative population of GPA (leukocytes) (Figure 2A). Then, CD45-H/SSC-H
analysis of CD45 and granularity was separated leukocytes into granulocytes, monocytes (R2
region) and lymphocytes (Figure 2B). The percentages and mean fluorescent intensity (MFI)
of CD16 and CD11b were analysis by histogram analysis (Figure 2C-D).
Statistic analysis
All descriptive analysis (mean, SD, coefficient of variation and ranges) was performed using
GraphPad PRISM 6.0 (GraphPad Software, Inc.). Comparisons between parameters were
analyzed with non-parametric Mann-Whitney U test. Simple linear regression and
Spearman’s correlation coefficient (rs) were calculated. The threshold for statistical
significance for all comparisons was chose as P < 0.05.
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Figure 1 Flow cytometric analysis of phagocytosis and oxidative burst of thalassemic
monocytes. Thalassemic whole blood sample was incubated with 50 μM DCFH-DA for 10
min at 37°C water bath, and then, co-incubated with 5×107 PI-fixed Escherichia coli stain
DH5α. Flow cytometric analysis shown (A) Leukocyte population was gated from GPA- cells
in R1 region. (B) FSC-H/SSC-H analysis of monocytes in R2 region. (C-I) Phagocytosis (PI,
x-axis) and oxidative burst (DCF, y-axis) of monocytes at time 0-180 min.
Figure 2 Flow cytometric analysis of CD16 and CD11b expression on thalassemic
monocytes. Thalassemic whole blood sample was lyzed RBCs and stained with
fluorochromes specific to surface markers. (A) Leukocyte population was gated from GPA-
cells in R1 region. (B) CD45-H/SSC-H analysis of monocytes in R2 region. Histogram
analysis of (C) CD16 and (D) CD11b expression on thalassemic monocytes was determined.
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Results :
Analysis of Escherichia coli phagocytosis by monocytes
The PI labeled E. coli was prepared. Fixed E. coli at 2×109 bacteria/mL was stained with PI
at 1, 10 and 100 μg/mL. The yield of PI-fixed E. coli was 100% positive in FL-3 channel
compared to unstained fixed E. coli (Figure 3). The mean fluorescent intensity (MFI) of PI
labeled E. coli increased with the increasing concentration of PI. The E. coli labeled with 100
μg/mL PI had higher MFI (1,635±81.2). This concentration was further used for phagocytosis
and oxidative burst activity study.
The amount of E. coli used for monocyte phagocytosis analysis was determined. Normal
whole blood samples (n = 3) were treated with different doses of PI-fixed E. coli (0.1-10×107
bacteria) at 90 min 37°C. The percentages and MFI of monocyte that phagocytose E. coli
were increased as dose-dependent manner (Figure 4). At 1-10×107 bacteria incubated with
monocytes had 22-3820% phagocytosis with increased MFI levels from 40±9 to 377±111.
The PI-fixed E. coli at 1×107
and 5×107
was used for phagocytosis and oxidative burst
activity study.
Figure 3 Flow cytometric analysis of propidium iodide labeled 70%ethanal fixed Escherichia
coli. (A) Histogram and (B) mean fluorescent intensity (MFI) of fluorescent in FL-3 channel
of fixed E. coli labeled with different doses of propidium iodide (PI) (1-100 μg/mL).
(Experiment repeated 5 times) *Significant different when compared to unstained fixed E.
coli (P < 0.05).
Figure 4 Dose effect of PI-fixed E. coli treated normal monocytes. (A) Percentages and (B)
mean fluorescent intensity (MFI) of phagocytosis on monocytes treated with 0.1-10×107
bacteria at 37°C water bath for 90 min.
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Impaired phagocytosis and oxidative burst of thalassemic monocytes associated to CD16
expression
LPS-primed monocytes can induce phagocytosis and oxidative burst of peripheral blood
monocytes to destroyed microbial infection. The phenomena of LPS tolerance and priming of
phagocytosis and oxidative burst activity of monocytes were found. Normal blood samples
were pre-treated with 1 and 10 ng/mL LPS before co-incubated with either 1×107 or 5×10
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PI-fixed E. coli (Figure 5A and 5B). Percentages of double positive of phagocytosis and
oxidative burst were increased in time dependent manner. Moreover, increased percentages
of double positive of phagocytosis and oxidative burst were found to be LPS-dose dependent
manner in 1×107 PI-fixed E. coli co-incubation (Figure 5-A1). The percentages of double
positive of phagocytosis and oxidative of 10 ng/mL LPS primed monocytes at 60, 90 and 120
min (19±11%, 30±9% and 36±7, respectively) were significant higher than unprimed
monocytes (3±1%, 23±10% and 23±9, respectively). However, the there was no effect of
LPS in monocytes treated with 5×107 PI-fixed E. coli (Figure 5-B1). Interestingly, the
percentages of oxidative burst in monocytes treated with 1×107 PI-fixed E. coli was increased
at 15 min in unprimed, 1 ng/mL and 10 ng/mL LPS-primed (38±4%, 58±6% and 68±21%,
respectively) and then drop down within 15 min later (1±1%, 1±1% and 1±2%, respectively)
(Figure 5-A3). At the same time of oxidative burst reduction, the percentages of phagocytosis
were increased up to 19±5% in all 3 conditions at 30 min and reached to plateau phase at 60
min (34±9%, 30±11% and 27±9%, respectively) (Figure 5-A2). The MFI of both
phagocytosis and oxidative burst in all conditions had similar trend to the percentages of
them (Figure 5-A4 and 5-A5).
Phagocytosis and oxidative burst functions of thalassemic monocytes were compared to
normal monocytes. At 5×107 PI-fixed E. coli co-incubation with both normal and thalassemic
monocytes, phagocytic and oxidative burst activities were tolerance to LPS stimulation
(Figure 5B and 5C). There was no significant different double positive of phagocytosis and
oxidative burst between unprimed and LPS-primed both normal and Thalassemic monocytes.
Unprimed normal monocytes had 3-fold higher phagocytosis than thalassemic monocytes at
30 min after PI-fixed E. coli co-incubation (35±11% and 11±5% phagocytosis, respectively)
(Figure 5-B2 and 5-C2). The maximum of phagocytosis in thalassemia was observed at
90 min after PI-fixed E. coli co-incubation (35±11% phagocytosis and 88±14 MFI) while
normal monocytes achieved the same phagocytosis level as patients within 30 min after
incubation (35±11% phagocytosis and 152±49 MFI). These results showed that thalassemic
monocytes were impaired phagocytosis and oxidative burst activity.
To address how function of thalassemic monocytes defect, serological parameters including
the levels of immunoglobulin (Ig) A, IgG and IgM were examined. Thalassemia patients in
this cohort study had normal levels of Igs (data not shown). Next, the expression of CD16
and CD11b were further examined by using flow cytometry (Figure 6). The percentages of
both CD16+ and CD11b
+ monocytes in thalassemia patients were no significant different
when compared to normal subjects. However, the level of CD16 expression on surface of
thalassemic monocytes both non-splenectomy and splenectomy was decreased significantly
compared to normal subjects (MFI 1,907±870, 1,873±572 and 2,976±982, respectively)
(P < 0.05). The Spearman’s correlation coefficient (rs) of the CD16 MFI and the percentages
of double positive phagocytosis and oxidative burst of monocytes were 0.750 at P < 0.05.
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Figure 5 Effect of LPS and incubation time on phagocytosis and oxidative burst of
monocytes in thalassemia. Whole blood samples from (A-B) normal subjects (n = 6) and
(C) non-splenectomized β-thalassemia/HbE (n = 5) were treated with (A1-A5) 1×107 and
(B1-B5 and C1-C5) 5×107 PI-fixed E. coli at 37°C water bath for 0-180 min.
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Figure 6 The CD16 and CD11b expression on Thalassemic monocytes. Peripheral blood
samples obtained from normal subjects (normal) (n = 12), non-splenectomized β-thalassemia/
HbE (β-Thal/E-NS) (n = 16) and splenectomized β-thalassemia/HbE (β-Thal/E-S) (n = 12)
were stained with florochromes conjugated specific to leukocyte markers and analyzed the
percentages and the mean fluorescent intensity (MFI) of (A) CD16 and (B) CD11b on
monocytes by using flow cytometer. *Significant different when compared to normal
subjects (P < 0.05).
Discussion and Conclusion :
Our three-color flow cytometric analysis of whole blood was effective and simple technique
for assessing phagocytosis and oxidative burst of phagocytes including monocytes and
granulocytes to bacterial infection in single tube. This technique is required only 440 μL of
whole blood and useful for detection in samples with the intact RBC or the immature RBC
contamination, for example, thalassemia blood samples. PI-label bacteria was simple
preparation and useful for the kinetic measurement of intracellular killing analysis.
Similarly, the impairment of monocytic phagocytosis in thalassemia has been documented in
previous studies (Wiener, 2003). The mononuclear phagocyte system is heavily involved in
the pathology of thalassemia by Fc-receptor-mediated clearance of defective RBCs that might
be affect to monocytic function. However, there was no strongly evidenced to document the
mechanism of monocytic dysfunction in thalassemia yet. In this study finds that loss of CD16
expression on monocytes was associated to impaired phagocytosis and oxidative burst
activity of thalassemic monocytes against to gram-negative bacteria. It might be contribute to
the increased susceptibility to infections
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Acknowledgements :
This study was supported by Thailand Research Fund (TRF) (MRG5980043, DPG5980001,
IRG5780009 and IRG5780011), Office of the Higher Education Commission and Mahidol
University under the National Research University Initiative, Research Chair Grant, National
Science and Technology Development Agency, Thailand and Faculty of Science and Faculty
of Medicine Ramathibodi Hospital, Mahidol University.
References
1. Bohmer, R.H., Trinkle, L.S., and Staneck, J.L. (1992). Dose effects of LPS on
neutrophils in a whole blood flow cytometric assay of phagocytosis and oxidative
burst. Cytometry 13, 525-531.
2. Fucharoen, S., and Winichagoon, P. (1987). Hemoglobinopathies in Southeast Asia.
Hemoglobin 11, 65-88.
3. Keawvichit, R., Khowawisetsut, L., Chaichompoo, P., Polsrila, K., Sukklad, S.,
Sukapirom, K., Khuhapinant, A., Fucharoen, S., and Pattanapanyasat, K. (2012).
Platelet activation and platelet-leukocyte interaction in beta-thalassemia/hemoglobin
E patients with marked nucleated erythrocytosis. Annals of hematology 91, 1685-
1694.
4. Morris, M.C., Gilliam, E.A., Button, J., and Li, L. (2014). Dynamic modulation of
innate immune response by varying dosages of lipopolysaccharide (LPS) in human
monocytic cells. The Journal of biological chemistry 289, 21584-21590.
5. Pattanapanyasat, K., Thepthai, C., Lamchiagdhase, P., Lerdwana, S., Tachavanich,
K., Thanomsuk, P., Wanachiwanawin, W., Fucharoen, S., and Darden, J.M. (2000).
Lymphocyte subsets and specific T-cell immune response in thalassemia. Cytometry
42, 11-17.
6. Vento, S., Cainelli, F., and Cesario, F. (2006). Infections and thalassaemia. The
Lancet Infectious diseases 6, 226-233.
7. Wanachiwanawin, W. (2000). Infections in E-beta thalassemia. Journal of pediatric
hematology/oncology 22, 581-587.
8. Weiss, G., and Schaible, U.E. (2015). Macrophage defense mechanisms against
intracellular bacteria. Immunological reviews 264, 182-203.
9. Wiener, E. (2003). Impaired phagocyte antibacterial effector functions in beta-
thalassemia: a likely factor in the increased susceptibility to bacterial infections.
Hematology (Amsterdam, Netherlands) 8, 35-40.
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