SHOCK, Vol. 21, No. 4, pp.

306310, 2004

ASSOCIATION OF INTERLEUKIN-10 PROMOTER POLYMORPHISM WITH

THE INCIDENCE OF MULTIPLE ORGAN DYSFUNCTION FOLLOWING
MAJOR TRAUMA: RESULTS OF A PROSPECTIVE PILOT STUDY
Ove Schroder, Reinhold Alexander Laun, Burkhard Held,
Axel Ekkernkamp, and Klaus-Martin Schulte
Department of Trauma Surgery, Ernst-Moritz-Arndt-University of Greifswald, and Unfallkrankenhaus
Berlin, Clinic for General Surgery and Trauma Surgery, Heinrich-Heine-University, Duesseldorf, Germany
Received 3 Jun 2003; first review completed 23 Jun 2003; accepted in final form 16 Jan 2004
ABSTRACTA dysbalanced immune response is thought to account for a substantial part of the morbidity and mortality
after severe trauma. The cytokine interleukin-10 (IL-10) suppresses the transcription of proinflammatory cytokines, mainly
in macrophages and monocytes. The objectives of this prospective study in a level I trauma center in Germany were to
examine the distribution of IL-10 promoter polymorphisms in a cohort of severely injured patients, to measure IL-10
cytokine levels and relate these to the genotype, and to identify associations of IL-10 polymorphisms with the incidence
of severe multiple organ dysfunction syndrome (MODS). The genotypes of polymorphisms 592 and 1082 were determined by polymerase chain reaction (PCR) and restriction cleavage with Rsa 1 or Mnl I, respectively. We analyzed 119
severely injured trauma patients [mean Injury Severity Score (ISS) 38.0 13.2]. The frequency of the 1082A allele was
0.542, and that of the 592C allele was 0.807. IL-10 polymorphisms were not significantly associated with mean systemic
IL-10 cytokine levels 6, 12, and 18 h after admission to the ICU. Carriers of the genotype 592AC had significantly higher
overall MOD scores than non-AC carriers (P = 0.018; Pcorr = 0.036). The genotypes of the IL-10 SNP 1082 were not
significantly associated with MOD scores. A multivariate Cox hazard regression analysis including important factors of the
patients anatomic and physiological trauma impact revealed only the shock index, the severity of the head injury, and the
IL-10 592 genotype AC as significant independent risk factors for the development of MODS. In this multivariate analysis,
carriers of the genotype 592AC displayed a 3.3-fold increase in the relative risk of developing a MODS (P = 0.008, hazard
ratio 3.29, 95% CI 1.367.97). Our data suggest a possible link between the AC genotype of the 592 single nucleotide
polymorphism and significantly higher mean MOD scores. The AC genotype was associated in multivariate analysis with
a higher relative risk of MODS in multiply injured patients. Further investigations in larger cohorts need to focus on the
potential diagnostic and therapeutic options of this SNP.
KEYWORDSIL-10, multiple organ dysfunction syndrome (MODS), polymorphism, cytokines, complication

INTRODUCTION

Recently certain polymorphisms in cytokine genes with transcriptional relevance in vitro have been identified. An increase
or decrease in cytokine production is associated with genetic
variations in the promoter region (1719). Polymorphisms
within the encoding region may lead to differences in activity
(10).
The human IL-10 gene was mapped to chromosome 1q31
32. The IL-10 5 flanking region contains numerous polymorphisms. Some of these directly influence protein expression
(17, 18, 20). There are three promoter single nucleotide polymorphisms (SNP) at position: 1082 (G to A substitution),
819 (C to T substitution), and 592 (C to A substitution) (21).
It is well established and has been confirmed in various
studies that IL-10 SNPs are completely (819 to 592) or
strongly (1082 to 819 and 592) linked. Consequently, in
the white population only three haplotypes were found: GCC
(G at position 1082, C at position 819, C at 592), ACC, and
ATA (22). These haplotypes have been associated with high
(GCC), intermediate (ACC), and low (ATA) IL-10 production
in several studies (17, 18). The highest IL-10 levels were
produced in individuals with homozygous GCC/GCC haplotypes (17, 18). The 592A allele shows a lower transcriptional
activity and consequently decreased IL-10 secretion in in vitro
studies (18, 22, 23).
IL-10 SNPs have been linked to various diseases. The
592A allele and the ATA haplotype (low IL-10 production)

Multiple organ dysfunction syndrome (MODS) is a frequent

and serious complication following trauma (1, 2). A dysbalanced immune response is thought to account for a substantial
part of the morbidity and mortality resulting from severe
trauma (36). Several studies have shown that severe trauma is
followed by massive cytokine production (710). Proinflammatory cytokines such as tumor necrosis factor (11) and
interleukin (IL)-6 (12) are important in the initial phase of
systemic inflammatory response syndrome (SIRS). These are
counterbalanced by anti-inflammatory cytokines such as transforming growth factor or IL-10, which are thought to reflect
the compensatory anti-inflammatory response syndrome
(CARS) (13).
IL-10 suppresses gene expression of proinflammatory cytokines (14,15) and has been shown to be elevated shortly after
trauma (16). IL-10 is associated with the development of
MODS following severe trauma, but the causal relationship
between these findings remains unclear (16).

Address reprint requests to Klaus-Martin Schulte, MD, Clinic for General and
Trauma Surgery, University Clinic of the Heinrich-Heine-University Duesseldorf,
Moorenstr 5, 40225 Dsseldorf, Germany.
This study was generously supported by the Hans-and-Gerti-Fischer-Stiftung,
Bochum, Germany. Klaus-Martin Schulte was supported by a personal grant from
the Deutsche Forschungsgemeinschaft (DFG Schu 1270/1-1).
DOI: 10.1097/01.shk.0000119239.88805.50

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INTERLEUKIN-10 PROMOTER POLYMORPHISM

are associated with severe asthma and sepsis (23, 24), whereas
haplotypes with high IL-10 production (GCC and the 592C
allele) are associated, e.g., with lupus nephritis (25).
In this prospective pilot study we examined the hypothesis
that the IL-10 promoter polymorphisms 1082 and 592 may
be relevant to the development of MODS in multiple trauma
patients.
MATERIALS AND METHODS
Patients
This prospective, observational study was approved by the institutional review
board for human research presented by the ethics committee of the Free University
of Berlin, Germany. Written informed consent was obtained from the patients or
next of kin. The trauma center UKB (Unfallkrankenhaus Berlin) fulfills the requirements of a level I trauma center as defined by the American College of Surgeons.
Study patients were treated as indicated by modern guidelines of trauma surgery,
anesthesiology, and intensive care medicine.
From May 1999 through August 2001, consecutive adult patients admitted to the
UKB for trauma were assessed for possible enrollment according to the inclusion
and exclusion criteria. The criteria for inclusion were presence of an initial Injury
Severity Score (ISS) above 15 combined with the presence of at least one lifethreatening injury and at least one additional severe injury in another part of the
body. All patients were diagnosed with an initial helical computed tomographic
study of the total body. Injury severity was assessed using the Injury Severity Score
(ISS) based on the Abbreviated Injury Scale (AIS) (26).
The physiological disarrangement on admission was assessed using the Acute
Physiology and Chronic Health Evaluation (APACHE II) score (on a scale from 0
to 71, with higher scores indicating more severe organ dysfunction (27).
The primary outcome measure was the development of MODS. It was defined
according to the MODS Score by Marshall (28) (status > 6 points) and calculated
as a single daily value during the ICU stay.
Blood specimens were collected in tripotassium EDTA sterile tubes and immediately stored at 80C. The preparation of genomic DNA was done as previously
described (10). DNA was stored at 20C for the analysis of the IL-10 polymorphisms.

Typing of IL-10 promoter polymorphisms

Typing of the IL-10 SNPs was performed by PCR on a Biometra thermocycler
(Whatman Biometra T Gradient, Goettingen, Germany). Oligonucleotide primers
for amplification were derived from published genomic sequences (Genbank
X78437.2). For the 592 SNP [Genbank (gi14625939) position 8700], a 412-bp
fragment was amplified with forward primer [5 GGT GAG CAC TAC CTG ACT
AGC 3 (position 85248244)] and reverse primer [5 CCT AGG TCA CAG TGA
CGT GG 3(position 89358916)]. The reaction mix for polymerase chain reaction
(PCR) contained 2575 ng DNA and 5 pmol oligonucleotides. Reactions were
performed in 8 L of a 1 solution of TaqMasterMix (Qiagen, Hilden, Germany).
Amplification was accomplished by initial incubation at 94C for 5 min followed by
36 cycles of denaturation at 94C for 30 s, annealing at 55C for 30 s, and extension
at 72C for 90 min. The reaction was completed by a final incubation at 72C for
10 min. This amplified a fragment of 412 bp containing a C/A polymorphism
generating an RSA I restriction cleavage site. For RFLP analysis of the IL-10
promoter gene, amplimers were digested by the restriction endonuclease RSA I at
37C in 1 buffer Y+ (Fermentas, Munich, Germany) for 24 h and subjected to
electrophoresis on 1.4% w/v agarose gels with subsequent staining by ethidium
bromide (1 g/mL). When an RSA I site was present, restriction digestion produced
two fragments of 176 bp and 236 bp, respectively. Size was determined by comparison to a molecular weight standard.
For the preparation of the 1082 SNP (GA) [Genbank (gi14625939) position
8211], a fragment was amplified with forward primer [5 CTC GCC GCA ACC
CAA CTG GC 3 (position 80998118)] and reverse primer [5 GCT TCT GTG
GCT GGA GTC 3 (position 83428325)]. Amplification of the fragment containing the 1082 AG polymorphism was done identically, except for the use of buffer
G+ (Fermentas, Munich, Germany) for the restriction digestion by Mnl I. Because
of the presence of the CA tandem repeat polymorphism in the amplified region, the
exact size of the undigested and digested amplimers was determined on sequencing
gels as previously described (29).

Statistical analysis
IL-10 allele frequencies were calculated by gene count. Continuous data are
expressed as mean standard deviation (SD). In case of proportions, the tests of
homogeneity between the groups formed by the genotype were calculated by the
chi-square test or Fishers exact test as appropriate. Comparison of the differences

AND

MULTIPLE ORGAN DYSFUNCTION

307

between the means of quantitative parameters was performed with the MannWhitney U test in case of two groups or with the Kruskal Wallis test in case of three
groups.
To estimate the univariate association of the IL-10 polymorphisms with the
incidence of MODS, we used the Kaplan-Meier method (30) and the log-rank test
to analyze statistical significance. This method was judged to be the most appropriate in the circumstances, i.e., severely injured patients who were in danger of
dying early as a result of factors that were not influenced by the genetic status. The
P values were corrected by the Bonferroni method according to the formula Pcorr
1 (1 P)n, where Pcorr is the corrected value, P is the uncorrected value, and n is
the number of loci.
Univariate analysis of other clinical factors that might influence the incidence of
MODS was derived from previous studies or the pathophysiological background.
The log-rank test was used for grouped and the Cox proportional hazards model for
continuous variables. All variables found univariately significant at P < 0.10 were
entered into a backward step-down Cox proportional hazard regression analysis
using a P < 0.05 criterion for interaction variable retention.
All statistical tests were two-sided. A P value < 0.05 was considered statistically
significant. Analyses were performed with SPSS for Windows (Version 11.5, SPSS
Inc, Munich).

RESULTS
Patient characteristics and gene frequencies

The analyzed cohort comprised a total of 119 consecutive

white severe multiple trauma patients. The baseline data of the
patients are shown in Table 1. The severely injured (mean ISS
38.0 13.2) patients were mostly young (34.3 17.4 years)
and showed a low background of preclinical morbidity.
Twenty-two (18.5%) patients developed a MODS (mean time
to MODS 4.4 3.5 days). Fifteen patients died within 24 h
(12.6%), and 28 (23.5%) within 90 days after trauma.
Trauma was caused by traffic accident (n 97), suicide
attempt (n 12), or other causes (n 10). The injury pattern
was characterized by severe trauma in all patients (ISS > 15).
Injury severity was almost equally distributed into four groups:
ISS 1529 (n 36), 3039 (n 31), 4049 (n 24), 50
(n 28).
The mean number of operations performed per patient was
3.5 3.2 (range 014). Substitution of erythrocytes was necessary in every patient (mean 5.4 5.2 L).
Table 2 shows the genotype distribution in our cohort.
Neither group deviated significantly from Hardy-Weinberg
TABLE 1. Baseline characteristics*
Characteristics
Age (yr.)
Male patients: no. of patients (%)
Anatomic injury
Injury Severity Score
Head injury (AIS points)
Thorax injury (AIS points)
Abdominal injury (AIS points)
Extremity injury (AIS points)
Baseline clinical and laboratory values
APACHE II
Shock Index
pH
Leukocytes (GPT/L)
More than 6 packages (330 mL each) of
erythrocyte concentrates in the first 24 h

All patients
(n = 119)
34.3 17.4
92 (77.3%)
38.0 13.2
3.0 1.9
3.3 1.3
1.9 1.7
2.2 1.1
20.8 5.4
0.89 0.30
7.32 0.11
11.1 4.8
47 (40.2%)

*Plus-Minus values are means standard deviation.

AIS, Abbreviated Injury Scale; APACHE, Acute Physiology and Chronic
Health Evaluation.

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TABLE 2. Frequencies of IL-10 promoter polymorphisms

1082 genotype
1082GG
1082AG
1082A
592 genotype
592CC
592CA
592AA

Polymorphism

24 (0.2)
61 (0.5)
34 (0.3)

IL-10 SNP at position 1082

GG vs. non-GG
AG vs. non-AG
AA vs. non-AA
IL-10 SNP at position 592
AA vs. non-AA
AC vs. non-AC
CC vs. non-CC

77 (0.6)
38 (0.3)
4 (0.03)

equilibrium (P 0.972 for the 1082 SNP, and P 0.984 for

the 592 SNP). The two SNPs at 1082 (G A) and 592 (C
A) are not randomly distributed (P < 0.0001). Only three out
of four possible combinations of the observed SNPs were
found in our population (GC, AC, and AA). Table 3 shows that
the patient groups formed by the genotype of the 592 SNP are
homogeneous in regard to trauma impact and important
preclinical and early clinical factors. The genotype groups of
the 1082 SNP were tested in equal proportions and did not
show any significant differences (data not shown).
IL-10 promoter polymorphisms and outcome

Table 4 shows the association of the IL-10 promoter genotypes with the incidence of MODS. There was no difference in
genotype distribution of the 1082. Patients carrying the genotype 592AC were overrepresented in the group of MODS (P
0.049, Pcorr 0.096, hazard ratio 2.2, 95% CI 1.06.4) as
compared with Non-AC-carriers. Figure 1 shows the KaplanMeier distribution of this association. Table 5 shows the maximal MOD scores across the genotype groups in each section of
the score system. Neurological scoring was not performed
because every patient was sedated. Carriers of the genotype
TABLE 3. IL-10 592 SNP: homogeneity of the genotype groups*
Parameter

CC
(n = 77)

Age 50 yr.
12
Female patients
19
Anatomic Injury Severity
ISS
37.7 14.3
Head Injury
(AIS points)
2.9 1.9
Thorax Injury
(AIS points)
3.4 1.4
Abdominal Injury
(AIS points)
1.7 1.7
Extremity Injury
(AIS points)
2.4 1.0
Physiological Injury Severity
APACHE II
20.8 5.2
Shock Index
0.90 0.33
Arterial pH
7.32 0.12
Leukocyte (GPT/L)
11.4 4.9
> vs. 6 packages of
erythrocyte
concentrates in the
first 24 h
43/32

ET AL.

No. of patients
(% of MODS
per group)

Hazard ratio
(95%
confidence
interval)

P
value

24 (8.3%) vs. 95 (21.1%)

61 (19.7%) vs. 58 (17.2%)
34 (23.5%) vs. 85 (16.5%)

0.4 (0.21.3)
1.3 (0.63.1)
1.3 (0.53.3)

.161
.528
.582

38 (28.9%) vs. 81 (13.6%)

77 (14.3%) vs. 42 (26.2%)

2.2 (1.06.4)
0.5 (0.21.2)

0.049
0.114

TABLE 4. IL-10 SNPs and MODS*

n (frequencies)
(n = 119)

Genotype

SCHRO DER

AC
(n = 38)

AA
(n = 4)

P
value

9
7

1
1

0.374
0.717

39.0 11.3

32.0 12.0

0.349

3.1 2.0

2.0 2.2

0.468

3.3 1.3

3.8 0.5

0.800

2.3 1.7

2.3 1.5

0.252

2.0 1.1

1.5 1.7

0.089

20.9 4.8
0.91 0.26
7.30 0.09
10.2 3.7

20.5 13.4
0.77 0.25
7.25 0.14
14.2 10.9

0.875
0.506
0.358
0.255

24/14

3/1

0.730

*All clinical values are determined within 2 h after arrival in the emergency room.
Plus-minus values are means standard deviation. P value is computed as an
overall comparison between the genotype groups with Kruskal-Wallis test.

Definition of abbreviations: ISS, Injury Severity Score; AIS, Abbreviated Injury

Scale; APACHE, Acute Physiology and Chronic Health Evaluation.

*We refrained from further analysis of the genotype 592 AA because of the small
number of patients (n = 4). P values and odds ratios were computed using KaplanMeier statistics and the log-rank test.

597AC had significantly higher overall MOD scores as

compared with nonAC carriers (P 0.018 ; Pcorr 0.036).
No other differences between any genotype and single-section
organ scores were statistically significant.
Following the finding of the strong connection between the
two SNPs, we analyzed whether any of the combinations of
genotypes were associated with the incidence of MODS. No
combinations of 1082 and 592 genotypes were significantly
associated with MODS.
To examine this finding in the complex clinical situation
after major trauma, we constructed a strongly time-dependent
regression model. First we analyzed various clinical factors
that were derived from the pathological background or previously published studies as related to the onset of MODS.
Results are shown in Table 6. The univariate analyses revealed
a significant association of (in order of significance) abdominal
injury, APACHE II, more than six packages of erythrocyte
concentrates in the first 24 h, head injury, arterial pH, and
shock index with MODS. Secondly, all factors with a level of
significance below a liberal P < 0.1 were included in a timedependent Cox hazard regression model, as described above.
The results are shown in Table 6. Only the shock index (P
0.002), the severity of the head injury (P 0.013), and the
IL-10 promoter polymorphism 592 genotype AC (P 0.008)
stayed significant.

FIG. 1. Cumulative incidence of MODS in the IL-10 592AC (n = 38)

versus non-AC (n = 81) genotype group (Kaplan-Meier estimation).

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INTERLEUKIN-10 PROMOTER POLYMORPHISM

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MULTIPLE ORGAN DYSFUNCTION

309

TABLE 5. IL-10 SNPs and maximal MOD-score values*

IL-10 SNP

MODS

Lung

Liver

Kidney

Circulation

Coagulation

All patients
1082
GG
AG
AA
592
CC
AC

4.4 2.5

1.4 1.1

0.9 1.1

0.4 0.7

1.7 1.3

1.5 1.1

3.9 2.0
4.5 2.5
4.7 3.1

1.0 1.0
1.4 1.2
1.5 1.0

1.0 1.2
0.8 1.0
0.9 1.3

0.2 0.4
0.5 0.8
0.4 0.6

1.9 1.3
1.8 1.3
1.5 1.3

1.4 1.0
1.5 1.2
1.6 0.9

4.2 2.5
4.9 2.9

1.3 1.1
1.6 1.2

0.9 1.1
0.9 1.2

0.4 0.6
0.5 0.9

1.8 1.6
1.6 1.2

1.4 1.1
1.6 1.1

*This table shows the maximum score values in the genotype groups (mean SD) according to Marshalls MOD score. All values were obtained in
the patient group alive 24 h after admission to the hospital. We refrained from further analysis of the genotype 592 AA because of the small number
of patients (n = 4).

IL-10 592 AC vs. non-AC genotype: P = 0.018; Pcorr = 0.036. There were no other significant differences between the mean score values of the
genotype groups.

DISCUSSION
The aim of the present study was to estimate the association
between SNPs in the IL-10 promoter region and MODS after
severe trauma. The patient cohort consisted of young patients
with a low background of coexisting morbidity. The described
genotype and allelotype frequencies were similar to previously
reported distributions in other studies (17, 22, 31). Because of
our use of strict criteria for defining MODS, the number of
outcome events was limited. We compared patients genotype
groups regarding baseline characteristics and trauma impact
and found no significant differences, which minimized the
influence of interfering factors.
A multiple organ dysfunction syndrome is a serious complication following trauma and is often related to increased
mortality (1, 2). Current evidence shows a causal relationship
between MODS and inflammatory dysregulation following
trauma (1214, 16). Current hypotheses stress the necessity for
a balance between the inflammatory (SIRS) and the antiinflammatory responses in the posttraumatic period. However,
TABLE 6. IL-10 592 genotype AC and MODS: time-dependent
univariate and multivariate analysis*

Parameter
IL-10 592 genotype AC
Age 50 yr.
Male patients
Anatomic injury severity
ISS
Head injury (AIS points)
Abdominal injury (AIS points)
Extremity injury (AIS points)
Physiological injury severity
APACHE II
Shock Index
Arterial pH
Leukocyte (GPT/L)
>6 packages of erythrocyte
concentrates in the
first 24 h

Univariate
analysis
P value
0.049
0.354
0.270
0.424
0.006
0.022
0.528
0.015
0.001
0.004
0.092

0.008

Multivariate Cox
hazard regression
analysis
Hazard ratio
(95% CI)

P
value

3.3 (1.47.9)

0.008

0.7 (0.60.9)

0.013
0.258

11.7 (2.555.4)

0.313
0.002
0.316
0.449

0.114

*All diagnostic and laboratory values were determined within 2 h of arrival in the
emergency room.
Definition of abbreviations: ISS, Injury Severity Score; AIS, Abbreviated Injury
Scale; APACHE, Acute Physiology and Chronic Health Evaluation; 95% CI, 95%
confidence interval.

many patients survive the initial SIRS status and are then
thought to be endangered by a state of T-cell hyporesponsiveness, suppression of T-cell proliferation, increase in T- and
B-cell apoptosis, and defects in antigen presentation, referred
to as compensatory anti-inflammatory response syndrome
(CARS) (14). IL-10 has been reported to reduce main components of the inflammatory process (14, 15, 3234). Back and
co-workers have shown in a murine model that the administration of antiIL-10 significantly increased the peak TNF
plasma levels after lipopolysaccharide (LPS) administration
(35).
Several studies have shown that the IL-10 production in
vitro is independently controlled by genetic polymorphisms in
the promoter region (17, 18, 22). These SNPs are completely or
strongly linked, a finding that is confirmed by our data (21).
The 592A allele has been shown to be associated with low
IL-10 concentrations and sepsis (23), whereas the 592C allele
is associated with higher IL-10 levels and lupus nephritis.
Our data support an impact of the IL-10 promoter polymorphism: 592AC influenced the extent of MODS after major
trauma. Although univariate analysis did not reveal a significant association with MODS after appropriate statistical
correction, the 592 AC genotype was significantly associated
with a 3.3-fold increase in risk to develop MODS. Small
sample size may be responsible for this phenomenon. A
cautious interpretation of these data is hence advised. Results
of a recently published study by Reid et al. point into a similar
direction. In a group of 88 older critically ill patients with
MODS, the frequency of in vitro high-producing IL-10 haplotypes was underrepresented in the MODS group as compared
with healthy controls (36). In our study the patients of the
genotype 1082GG were also less frequently in the MODS
group, but this lacks statistical significance, which again is
possibly because of the small sample size.
Previous studies have shown that an excessive inflammatory
reaction puts the patient at risk of developing organ insufficiency and/or MODS (37, 38). In an animal model, Welborn et
al. examined the influence of endogenous IL-10 production on
local and distant organ failure after visceral ischemia
reperfusion injury in wild-type and IL-10(/)-null C57BL/6
mice. They found that the magnitude of the lung tissue proinflammatory cytokine response, neutrophil infiltration, and
injury were greater in the IL-10(/)-null mice. On the other

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SHOCK VOL. 21, NO. 4

hand, exogenous IL-10 administration resulted in a decrease in

neutrophil infiltration in the IL-10(/)-null mice (39).
Carriers of the 592A allele have been shown to produce
less IL-10 (18, 22). In combination with the results of the
described animal model, a genetically influenced, locally
differentiated IL-10 expression is a possible pathological link
between the association of the genotype 592 AC and a higher
risk of MODS. Measurement of local IL-10 production in vivo
under circumstances of trauma is needed to finally validate this
argument. In summary, this study presents an association
between the IL-10 promoter polymorphism 592AC and the
incidence of MODS.

SCHRO DER

17.

18.

19.

20.

21.

ACKNOWLEDGMENT

22.

The authors would like to thank Prof. Dr. K.-E. Biebler, Dr. B. Jaeger, and Dr.
M. Wodny (University of Greifswald, Germany) for advice on statistical analysis.
The authors thank the skillful assistance of Mrs. K. Alemazkour in analysis of the
polymorphisms.

23.

24.

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