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Comparative proteomic studies of plasma from children with pneumococcalpneumoniaMing-Han Tsai ab; Tzou-Yien Lin cd; Sen-Yung Hsieh ce; Chih-Yung Chiu ab; Cheng-Hsun Chiu cd; Yhu-CheringHuang cd

a From the Department of Pediatrics, Chang Gung Memorial Hospital, Keelung b Graduate Institute of ClinicalMedical Sciences, Chang Gung University, Taoyuan c College of Medicine, Chang Gung University, Taoyuand Division of Pediatric Infectious Diseases, Department of Pediatrics, Chang Gung Children's Hospital,Taoyuan e Clinical Proteomics Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan

First Published:2009

To cite this Article Tsai, Ming-Han, Lin, Tzou-Yien, Hsieh, Sen-Yung, Chiu, Chih-Yung, Chiu, Cheng-Hsun and Huang, Yhu-Chering(2009)'Comparative proteomic studies of plasma from children with pneumococcal pneumonia',Scandinavian Journal ofInfectious Diseases,41:6,416 — 424

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ORIGINAL ARTICLE

Comparative proteomic studies of plasma from children withpneumococcal pneumonia

MING-HAN TSAI1,2, TZOU-YIEN LIN3,4, SEN-YUNG HSIEH3,5, CHIH-YUNG CHIU1,2,

CHENG-HSUN CHIU3,4 & YHU-CHERING HUANG3,4

From the 1Department of Pediatrics, Chang Gung Memorial Hospital, Keelung, 2Graduate Institute of Clinical Medical

Sciences, Chang Gung University, Taoyuan, 3College of Medicine, Chang Gung University, Taoyuan, 4Division of Pediatric

Infectious Diseases, Department of Pediatrics, Chang Gung Children’s Hospital, Taoyuan, and 5Clinical Proteomics Center,

Chang Gung Memorial Hospital, Taoyuan, Taiwan

AbstractStreptococcus pneumoniae is the primary pathogen causing community acquired pneumonia in children. Despite medicalprogress, the prevalence of complicated pneumococcal pneumonia became increased without apparent explanations. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to compare the plasma protein profiles fromchildren with different severities of pneumococcal pneumonia. Plasma samples from 14 cases, 7 with complicated and theother 7 with uncomplicated pneumonia, were analyzed. Complicated pneumonia was defined by the presence of pleuralfluid parameters consistent with empyema, and/or a computed tomography compatible with necrotizing pneumonitis. Thenormal control group included 7 age-matched volunteers. By comparing the plasma proteins of patients with differentseverities, 4 proteins with significant differences were identified. The up-regulated proteins were haptoglobin andimmunoglobulin kappa chain. The down-regulated were apolipoprotein A-I (Apo-AI) and transthyretin. All these proteinsare known to take part in the inflammation reaction, which implicates the active innate immune responses in severeinfections of S. pneumoniae. In addition, the up-regulated haptoglobin, which protects lung tissues against oxidativedamage by the clearance of hemoglobin, can also act as an inflammatory inhibitor. Thus, our data seem to indicate thatinflammation balance may take place in the occurrence of complicated pneumococcal pneumonia.

Introduction

Streptococcus pneumoniae is the most common

cause of bacterial pneumonia in children [1,2]. The

formation of pneumatoceles or necrosis had been

previously considered a rare complication in children

with pneumococcal pneumonia. However, since the

1990s, an increase in the incidence of complicated

pneumonia has been observed among children,

especially in Taiwan, and the cause of this increase

is not well understood [1,3�6].

There have been many studies on S. pneumoniae

itself, but few studies were performed on the

mechanisms in vivo in the affected patients [7,8].

Plasma is a widely used clinical resource that is

closely correlated with disease progression, and

analysis of the plasma protein profile alterations

in affected children is a promising way to elucidate

the mechanisms of complicated pneumococcal

pneumonia [9].

Proteomics, a large-scale study of gene expres-

sion at the protein level, is a rapidly developing area

of biomedical research [10]. Many studies have

demonstrated that determination of protein profiles

is useful in elucidating pathological pathways as well

as alterations associated with disease processes [11].

With the development of clinical proteomics, many

technologies have been employed to study plasma

proteomes [12]. In the present study, we used a

combination of techniques, including 2-dimensional

gel electrophoresis (2-DE), image analysis, and

mass spectrometry (MS), to compare the plasma

protein profiles among pneumococcal pneum-

onia children with different severities and hea-

lthy individuals. Potential markers associated with

Correspondence: T.-Y. Lin, Division of Pediatric Infectious Diseases, Department of Pediatrics, Chang Gung Children’s Hospital, No. 5 Fu-Hsin Street,

Kweishan 333, Taoyuan, Taiwan. Tel: �886 3 3281200, extn. 8002; Fax: �886 3 3288957. E-mail: pidlin@adm.cgmh.org.tw

Scandinavian Journal of Infectious Diseases, 2009; 41: 416�424

(Received 28 December 2008; accepted 30 March 2009)

ISSN 0036-5548 print/ISSN 1651-1980 online # 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis As)

DOI: 10.1080/00365540902936909

Downloaded By: [Chang Gung Memorial Hospital] At: 07:32 25 June 2009

complicated pneumococcal pneumonia were also

identified.

Materials and methods

Patient enrollment and sample preparation

This study was approved by Institutional Review

Board of Chang Gung Memorial Hospital. From

January 2002 to June 2005, patients less than 18 y of

age with pneumococcal pneumonia were enrolled

into our study. Those with concomitant life-threaten-

ing diseases or immunodeficiency were excluded.

The study population was categorized into 3 groups

in accordance with the disease severity: group 1

(severe test group), patients with complicated pneu-

monia; group 2 (mild test group), patients with

uncomplicated lobar pneumonia; and group 3 (con-

trol group), children visiting our outpatient depart-

ment (OPD) for allergen survey without any

infectious diseases. We collected plasma specimens

on the first d of admission. To remove albumin and

immunoglobulin (IgG) from human plasma, the

samples were treated with a Proteoprep Immunoaffi-

nity Albumin and IgG Depletion Kit (SIGMA)

according to the manufacturer’s instructions.

Pneumonia was defined as the combination of

acute respiratory symptoms and a consolidation on

chest radiographic images, which were interpreted

by attending physicians and radiologists. Compli-

cated pneumonia was defined by the presence of

pleural fluid parameters consistent with empyema

(based on visual or biochemical characteristics) and/

or cavitations or pneumatoceles on a computed

tomographic image compatible with necrotizing

pneumonitis [1]. The diagnostic criteria of pneumo-

coccal infection were a culture (including blood or

pleural effusions) yielding Streptococcus pneumo-

niae or a positive result of pleural pneumococcal

antigen.

Two-dimensional electrophoresis (2-DE)

500 mg of plasma were diluted to 330 ml with

rehydration solution (8 M urea, 4% CHAPS,

50 mM DTT, 0.2% v/v (pH 3�10) IEF buffer, trace

bromophenol blue). The samples were loaded on

immobilized pH gradient gel strips (pH 3�10), and

isoelectric focusing (IEF) was performed using a

Protean IEF Cell system (BioRad). The gels were

rehydrated overnight at 50 V. Proteins were focused

subsequently for 1 h at 100 V; then a gradient was

applied from 100 to 8000 V for 1 h and finally at

10,000 V to give a total of 55,000 Vh. All IEFs were

carried out at 208C. After the first-dimensional IEF,

IPG strips were placed in an equilibration solution

(6 M urea, 2% SDS, 30% glycerol, 0.375 M Tris-

HCl, pH 8.8) containing 2% DTT and were shaken

for 20 min at 50 rpm in an orbital shaker. The strips

were then transferred to the equilibration solution

containing 2.5% iodoacetamide and shaken for

another 20 min before being placed on a 12.5%

polyacrylamide gel slab. Separation in the second

dimension was carried out using Protean II electro-

phoresis equipment. Afterwards, the gels were fixed

and SYPRO Ruby stained.

Protein visualization and image analysis

For SYPRO Ruby staining, gels were initially fixed for

60 min in a buffer containing 10% methanol and 7%

acetic acid. The gels then were stained for 4 h in a

commercially available SYPRO Ruby buffer (Mole-

cular Probes, Eugene, OR) followed by washing in the

fixation buffer. Protein patterns in the gels were

recorded as digitalized images using a high-resolution

scanner (ProExpress, PerkinElmer, Boston, MA).

The SYPRO Ruby stained gels were used for image

analysis using the Progenesis software package (Pro-

genesis Discovery, Nonlinear Dynamics, Durham,

NC).

Protein analysis by mass spectrometry

The spots of interest were manually excised

from the gels. Proteins selected for analysis were

in-gel digested with trypsin. Briefly, the spots were

destained using 50 mM NH4HCO3 in 50% ACN

and dried in a SpeedVac concentrator. Subsequently

the samples were digested with trypsin (Promega,

Madison, WI) at 0.002 mg/ml in 25 mM NH4HCO3

(pH 8.5). After digestion, the supernatant was

collected and 1.5 ml was spotted onto a matrix-

assisted laser desorption/ionization (MALDI) target

plate.

Measurements were performed using the MALDI

time-of-flight mass spectrometry (Ultraflex; Bruker

Daltonics, Germany) and its settings were as follows:

ion source 1, 25.0 kV; ion source 2, 22.15 kV; lens,

6 kV; reflector, 26.3 kV; reflector 2, 14.65 kV; pulse

ion extraction, 120 ns. Ionization was achieved by

irradiation with a 337-nm nitrogen laser (l�337 nm)

and the high purity nitrogen gas (99.999%) was

maintained at 2050 mbar. The laser was operated at

25 Hz with laser power of 30%. For matrix suppres-

sion, we used a high gating factor with signal

suppression up to 400 Da. The mass calibration

was executed with the peptides in a mass range of

760�3100 Da. For each MALDI spot, spectra were

accumulated to the intensity of 2.5�10�4 arbitrary

intensity. We then used the flexAnalysis 2.0 and

BioTools 2.2 (Bruker Daltonics, Germany) to process

Plasma proteome in complicated pneumococcal pneumonia 417

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the raw data and search the processed peak lists via

MASCOT software 2.1 (in-house version) from

matrix science (www.matrixscience.com) to identify

the proteins.

Enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA)

was used to detect the antibodies to the recombinant

protein antigens. Briefly, 100 ml of the diluted

antigens (2�5 mg/ml) in PBS buffer, pH 7.4, was

added to the wells of the flat-bottomed ELISA

plates, and incubated overnight at 48C in a moist

chamber. The wells were then washed with a PBS

solution, pH 7.4, containing 0.05% Tween 20

(PBST) and blocked by the addition of 200 ml

PBST-0.1% BSA at room temperature for 1 h. After

emptying the wells, 100 ml sera with serial 2-fold

diluted in PBST-1% BSA was added to the wells,

which were then incubated at room temperature for

2 h. After washing the wells, 100 ml of the strepta-

vidine conjugated to horseradish-peroxidase diluted

1:5000 in PBST-1% BSA was added and the

incubation was continued at room temperature for

1 h. After an additional wash, 100 ml of the substrate

solution (TMB(tetramethylbenzidine)) was added to

the wells, which were then incubated at room

temperature for approximately 15 min. The reaction

was then stopped with 50 ml 2.0 N H2SO4 and the

absorbance was read at 450 nm. Each test was

performed in duplicate, and the mean absorbance of

the wells with no antigen was subtracted from that of

the wells with the protein antigens prior to analysis.

The criteria for sensitivity were determined by

adding 2 SD to the mean absorbance in the sera

from the healthy controls in this study.

Western blot analysis

Each plasma sample was loaded with the same

quantity. Protein from plasma was electrophoresed

on a 12% SDS-polyacrylamide gel and then trans-

ferred onto PVDF membrane. Actin was used as a

reference (internal control). The membranes were

blocked 1 h at 378C in 20 mM Tris-base, 150 mM

NaCl, pH 7.4, 0.05% Tween-20 (TBST) containing

10% skimmed milk. The primary antibodies used

were anti-fetuin polyclonal antibody or anti-alpha-1

anti-chymotrypsin (ACT). Membranes were incu-

bated overnight at 48C with each primary antibody,

and then were washed 3 times with TBST and

incubated with horseradish peroxidase-conjugated

secondary antibody for 1 h at room temperature.

Visualization of the immunoreactive proteins was

accomplished using ECL reagents. All the mem-

branes were exposed on the same X-ray film; then, 1

semi-quantitative analysis based on OD was

performed for Western blot films by Bio 1D��software (Vilber Lourmat, France).

Statistical methods

Data were analyzed with the SPSS Statistical Pack-

age (version 11.0). Fisher’s exact test was used to

analyze categorical data, and the Wilcoxon rank-sum

test or ANOVA was used to analyze continuous data.

A p-valueB0.05 was considered to be statistically

significant.

Results

Comparative proteomic analysis using 2-DE

Considering individual variability, we chose cases

with similar age distribution in each experimental

group and there were 7 cases in each group. The

average timing of sample collection was 8.594.2 d

after onset of disease. Table I shows the clinical

features of our patients. There were no significant

differences in the demographic and laboratory data

among the 3 groups. However, all cases in group 1

(complicated pneumonia cases) were admitted to the

pediatric intensive care unit (PICU) and received

invasive procedures (i.e. chest tube drainage or

operation), but in group 2 (uncomplicated lobar

pneumonia cases), only 1 case received invasive

procedure and neither of them was admitted to

PICU during hospitalization.

2-DE was used to study the alterations of plasma

protein profiles among the 3 study groups. The

representative 2-DE images are shown in Figure 1A.

More than 400 protein spots were detected on the

2-DE gels, which localized in the ranges of pI 3�10

and Mr 10�200 kDa. The 2-DE images were

analyzed by Progenesis 2D Elite software to esti-

mate the abundance of proteins in each sample and

to generate quantitative data. Based on a threshold

ratio of 2.0, the software detected the spots of

interest that revealed a significant change among the

3 groups. Protein spots that could not be identified

were excluded. Four different protein spots, includ-

ing 2 up-regulated and 2 down-regulated spots in

plasma of complicated pneumonia children, were

observed showing differences between the 3 experi-

mental groups (Figure 1B).

Protein identification

After scanning, the gels were stained with SYPRO-

Ruby and 4 protein spots with significant change

were picked using a SpotCutter and then subjected

to in-gel digestion for MALDI-TOF MS analysis.

418 M.-H. Tsai et al.

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MALDI-TOF MS was then used to analyze the

peptides after in-gel digestion. Table II reveals the

identified proteins, including Swiss-Pro accession

number, protein description, protein coverage, iden-

tification method, score, fold changes and functional

classification. The changes in the identified proteins

in 3 different groups can be summarized into 2

modes: 1) haptoglobin and immunoglobulin kappa

chain C region, which were up-regulated in the

group with complicated pneumonia compared to the

other 2 groups; and 2) apolipoprotein A-I (Apo-AI)

and transthyretin, which were down-regulated in the

group with complicated pneumonia compared to the

other 2 groups.

Validation of the up-regulated expression proteins and

down-regulated expression proteins by ELISA and

Western blot

To validate the results of our study, ELISA and

Western blot were used for further confirmation of

4 differential proteins. Initially, we investigated the

seroreactivity of 4 differential proteins using ELISA

and the relative concentration levels of these pro-

teins are shown as Table III and Figure 2. The

ELISA results verified the significant up-regulation

of haptoglobin/immunoglobulin kappa chain and

down-regulation of Apo-AI/transthyretin in group 1

(complicated pneumonia group). We selected 2

differentially expressed proteins, haptoglobin and

Apo-AI, for further confirmation using Western blot

analysis. Figure 3 shows the scanned Western blot

films (Figure 3A, 3B). The OD of each band in the

film was evaluated by Bio1D�� software (Vilber

Lourmat, France) (Figure 3C). Compared to that

derived from normal control group and cases with

uncomplicated lobar pneumonia, up-regulation of

haptoglobin and down-regulation of Apo-AI in the

sera of complicated pneumonia group were verified.

Discussion

In this study, we used the comparative proteomics

technology, 2-DE and MS, to demonstrate the

characteristic changes of the plasma proteins in

different severities of pneumococcal pneumonia.

Several plasma proteins altered significantly in cases

with complicated pneumococcal pneumonia. The

up-regulated proteins were haptoglobin and immu-

noglobulin kappa chain C region, and the down-

regulated proteins were Apo-AI and transthyretin.

All of the altered proteins are acute phase proteins

(APPs) and known to take part in the inflammation

reaction [13].

Like other acute respiratory infections, S. pneumo-

niae can induce the activation of immune response,

and this activation may cause lung tissue damage,

airway occlusion and then extravasation of erythro-

cytes into the lower respiratory tract [9,14,15]. Free

iron released from hemoglobin after hemolysis can

catalyze the formation of reactive oxygen species and

lead to oxidative damage in lung tissue [16]. Hence it

is very important to keep the balance of the immune

system between the ability to clear the pathogen and

the damage to the delicate structure of lungs [9].

Haptoglobin, 1 of the up-regulated proteins found in

our complicated pneumococcal pneumonia patients,

is a member of the acute-phase reactants [15]. It has

long been known as a major hemoglobin-binding

protein associated with hemoglobin catabolism [17].

Plasma haptoglobin could contribute significantly to

the clearance of hemoglobin and thus protect the

lower respiratory tract against hemoglobin mediated

oxidative damage [15]. Another important biologic

function of haptoglobin is the modulation of the

immune response [15]. In a previous study, hapto-

globin has been shown to decrease the reactivity of

lymphocytes and neutrophils toward a variety of

stimuli, and may act as a natural antagonist for

receptor-ligand activation of the immune system

Table I. Clinical characteristics of selected children analyzed with proteomic methods stratified by different severities of pneumococcal

pneumonia.

Parameters Group 1 n�7 complicated Group 2 n�7 lobar Group 3 n�7 control p-valuea

Age (months) mean9SD 34.3911.7 44.499.1 46.3910.8 0.102

Gender (male/total) 2/7 5/7 5/7 0.174

WBC (/mm3) mean9SD 16371.497763.1 17700.098644.1 � 0.767

Segment (%) 75.798.4 67.1919.2 � 0.314

CRP (mg/l) mean9SD 173.0968.0 175.09133.7 � 0.972

Operation 7/7 0 � 0.001

Chest tube 7/7 1/7 � 0.005

PICU admission 7/7 0 � 0.001

WBC: white blood cell; CRP: C-reactive protein; SD: standard deviation; PICU: pediatric intensive care unit.

Data presented as no/total or mean9standard deviation.apB0.05 was considered to be statistically significant; Fisher’s exact test and Wilcoxon rank-sum test used for analysis.

Plasma proteome in complicated pneumococcal pneumonia 419

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[18]. Thus, neutrophil respiratory burst activity, like

the occurrence of complicated pneumococcal pneu-

monia, can be inhibited by haptoglobin, suggesting

protective roles of haptoglobin in lung tissues.

Two down-regulated proteins, Apo-AI and trans-

thyretin, belong to the negative APPs that play major

roles in the transport of nutrients, hormones and

metabolites [9]. Apo-AI is a major component of

Figure 1. (A) (Left) Two-dimensional gel of plasma from 7 control volunteers using a pH range of 3 to 10. Four labeled areas were analyzed

in each group of subjects. (Right) Representative 2-dimensional electrophoresis gel images of plasma from children with complicated

pneumococcal pneumonia, uncomplicated lobar pneumococcal pneumonia, and age-matched control subjects using a pH range of 3 to 10.

(B) Focal 2-dimensional gel images demonstrate the differential expression of 4 identified proteins among the 3 study groups.

420 M.-H. Tsai et al.

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high-density lipoproteins (HDL) [19]. Recently,

lipoproteins have been found to play a key role in

innate immunity, and changes in lipoprotein levels

have been reported in a variety of inflammatory

disorders [20]. Of all lipoproteins, HDL has the

highest lipopolysaccharide (LPS, a component of

the outer membrane of Gram-negative bacteria)

and lipoteichoic acid (LTA, a major cell wall

component of Gram-positive bacteria) binding

capacity [21,22]. In vivo and in vitro experiments

have shown that bacterial substances such as LPS

and LTA are neutralized by HDL or Apo-AI,

suggesting anti-inflammatory properties of HDL

[21�23].

Our study revealed that the concentrations of

Apo-AI, the major component of HDL, were

markedly reduced and the changes were closely

related to the severity of the pneumococcal

pneumonia. The diminution of HDL in severe

infections has been well known, but the mechan-

isms are not well understood [20,24]. One possi-

bility is that HDL is consumed significantly by

binding bacterial substances [21,22]. Another

possibility is that high concentrations of proin-

flammatory cytokines during sepsis suppress HDL

production and facilitate HDL degradation

[25,26]. The decline in HDL renders the host

tissue more susceptible to bacterial substance-

mediated injury, resulting in cytokine overproduc-

tion and a further depletion of HDL (vicious

circle) [24]. Further prospective, randomized

designed studies are needed because it remains

unclear whether a lower Apo-AI level in children

with complicated pneumococcal pneumonia is

only an epiphenomenon or has a causal relation-

ship with adverse clinical outcomes.

Although our results cannot completely resolve

‘‘the cause of the increase of complicated pneu-

mococcal pneumonia in Taiwan’’, we still derived

the important concept that a defense homeostasis

may occur during the progression of complicated

pneumococcal pneumonia. This phenomenon is

not only specifically found in complicated pneu-

mococcal pneumonia, but also found in other

severe pathogen-infectious pneumonia such as

SARS coronavirus-associated pneumonia [9].

Thus, we can utilize these identified proteins as

markers to predict the clinical outcomes, espe-

cially during the treatment of severe respiratory

tract infection.

In conclusion, we reported an application of

proteomic approaches to elucidate the possible

pathological process for complicated pneumococcalTab

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Plasma proteome in complicated pneumococcal pneumonia 421

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pneumonia in childhood. Our studies demonstrate

that all of the altered proteins found in complicated

pneumococcal pneumonia children are APPs. They

are known to take part in the inflammation reaction

[13], which implicates the active innate immune

responses in severe infections with S. pneumoniae.

Due to the protective role of up-regulated haptoglo-

bin, our results also showed that there is some

Table III. Seroreactivity of 4 proteins among 3 study groups by ELISA testing.

Study group Complicated (mean9SD) Uncomplicated (mean9SD) Control (mean9SD) p-value

Haptoglobin (mg/ml) 0.73490.259 0.59390.296 0.24290.157 0.007

Apolipoprotein A-I (mg/ml) 4135.991763.1 4696.291829.7 6629.293583.7 0.208

Immunoglobulin kappa chain C region (mg/ml) 3.97690.877 3.21790.781 2.08590.621 0.001

Transthyretin (Prealbumin) (mg/ml) 2.53391.210 3.10691.751 7.01793.788 0.007

SD: standard deviation.

ANOVA was used to compare the mean levels of 4 proteins among the 3 study groups.

Figure 2. Seroreactivity of 4 proteins among 3 study groups by ELISA testing shown as scatter plots.

422 M.-H. Tsai et al.

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mechanism that sustains the inflammation balance

in the occurrence of complicated pneumococcal

pneumonia.

Acknowledgements

We thank the members of the Clinical Proteomics

Center of Chang Gung Memorial Hospital,

Taoyuan, Taiwan, for excellent technical assistance.

This work was supported by a grant from Chang

Gung Memorial Hospital (CMRPG 33061).

Declaration of interest: The authors report no

conflicts of interest. The authors alone are respon-

sible for the content and writing of the paper.

Figure 3. Western blot results validating the increased expression of haptoglobin in the group with complicated pneumococcal pneumonia

(A). The decreased expression of apolipoprotein in the group with complicated pneumococcal pneumonia (B). Actin was used as a

reference; the semi-quantitative analysis of Western blot films based on the OD of bands (C).

Plasma proteome in complicated pneumococcal pneumonia 423

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