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To understand the nature of immune dysfunction in patients with sepsis mani-
festing after severe trauma, a robust whole blood ex vivo test system (TruCul-
ture
TM
) has been developed.Methods: One milliliter of blood was drawn into sepa-
rate syringes containing ligands to stimulate the following TLR2-9. Amongst many
IL-
1ratio and TNFratio
] /sTNF-RII[pg/Ml], respectively.
Results: When compared with healthy donors, most TLR induced cytokines were
lower in trauma and even lower in sepsis patients’ cultures. An exception was the
-
tokines as well as soluble receptors in trauma and sepsis than in healthy donors.
Among the other TLR responses, TLR3 was most dramatically downregulated in
patients with trauma and even more in sepsis patients. Calculating the IL1ratio
and the TNFratio
Healthy donors had a median IL1ratio of 1.48 and a median TNFratio of 2.73,
trauma patients had 10times -, and sepsis patients had 100times lower IL1 and
TNFratios. Conclusion: The here developed TruCulture
TM
ex-vivo whole blood
-
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cally established stages trauma and sepsis. Results may substantially contribute to
signalling pathways leading to immune dysfunction.
0U[YVK\J[PVU
-
and hypotension, and may impair organ functions. The severe form of an initial insult
-
scribes SIRS followed by the presence of immunologically uncontrolled pathogens
or their toxins[1]. Many clinical parameters have been measured, and, also, several
potential markers have been suggested to describe the transitions during the disease
course[2-4]. Combining clinical outcome, proteomics as the important element of a
system biology approach enables an integrative outlook of a complex nature of signal-
ing networks in sepsis and may help identify critical regulatory nodes for therapeutic
manipulation[5,6]. One approach is to restimulate patients’ blood cells via their Toll-
like-receptors (TLR) representing the pattern recognition receptors of the immune
system. TLR ligands constitute the most important molecular structures called PAMPs
(pathogen-associated molecular patterns) (PAMPS)[7,8].
7H[PLU[Z
Sixteen ICU postoperative/posttraumatic trauma (SIRS) or sepsis patients were
included in this prospective pilot study and recorded daily over 4–15 days. From
admission to ICU to discharge from ICU, the assessment of clinical data was based
-
cal variables, age, type of admission (medical and scheduled/unscheduled surgery),
cancer and hematologic malignancy) [9] for severity of disease, and SOFA (Se-
quential Organ Failure Assessment) Score (status of 6 organ systems (respiration,
coagulation, liver, cardiovascular, central nervous system, renal)[9] for severity of
organ dysfunctions.
years.Male to female ratio was 4/5. The median SAPSII was 25 (range 16 to 32).
The median SOFA was 5 (range 1 to 15).
to female ration was 6/1. The median age was 58 years (range 42-76 years). The
median SAPSII was 36 (range: 15 to 43). The median SOFA was 5 (range 0 to 8).
ranged from 18 – 53.
4HYJO [O[O4\UPJO.LYTHU`
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The TruCulture
TM
system (www.rulemsbasedmedicine.com) isdesigned to draw
one milliliter of blood was drawn into separate syringes each of which contained
just one ligand to stimulate the following TLRs: TLR1/2(MALP); -2/6(Pam3Cys),-
The biomarkers secreted by blood cells as well as soluble receptors released from
(www.rulesbasedmedicine.com). The parameters IL1ratio and o following
GraphPadPrism™ 5 was used to create graphs and calculate differences between
groups using Mann-Whitney-U tests.
9LZ\S[ZKPZJ\ZZPVU
-
dardized assay (TruCulture
TM
-
duced by trauma alone or associated with a major infectious complication (sepsis or
septic shock) alters the capacity of the immune system to respond to an additional
stimulus.
Figure 1 shows that blood sampling into TruCulture
TM
tubes is easy and can be
accomplished in less than 90 seconds per patient. Supernatants can be harvested in
a bedside fashion and may be shipped for multiplexed determination of biomarker
concentrations to an adequate research laboratory.
Figure 2 shows the spontaneous release of IL-1ß, TNF-α, IL-1RA and sTNF-RII
determined in culture supernatants w/o stimulant. With the exception of the shock
sTNF-RII. One may interprete in vivo activation by the analysis of spontaneous
α and its binding molecules, the higher
release of antagonists in sepsis patients may be explained by the (compensatory)
been further elaborated by others [1,10-12].
;9(<4(:/6*205-3(44(;065(5+:,7:0:¶;:0:
Ex Vivo Simulation of Infection Using TLR- Whole Blood Stimulation (ICU)
IL-1ß w/o stimulus
Healthy Trauma Sepsis
0
1
2
3
4
5
6
7
8
pg/mL
IL-1RA w/o stimulus
Healthy Trauma Sepsis
0
1000
2000
3000
4000
pg/mL
TNF-D w/o stimulus
Healthy Trauma Sepsis
0
10
20
30
pg/mL
sTNF-RII w/o stimulus
Healthy Trauma Sepsis
0
10000
20000
30000
40000
pg/ml
Fig 1 -
TM
!" #$%
#&'%() *
Fig 2 - +,!$-./$0,!$"1./$",,
TM
%
*
4HYJO [O[O4\UPJO.LYTHU`
were recorded and are summarized in Figure 3. When compared to healthy donors,
-
lowing TLR2/6 (Pam3Cys) and TLR2/1 (FSL-1) stimulation (Figure 3). However,
-
-
stimulation by ligands binding to TLR4 has been described previously[13]. This
context may explain why sepsis patients’ blood cells have a reduced response to
increased TLR2 responsiveness as observed in our trauma patient population has
-
cytokines as well as endotoxin to upregulate the TLR2 expression and its response.
The generally lower cytokine responsiveness of patients with sepsis/septic shock
following TLR3, -4, and -5 stimulation is not explained by a lower surface expres-
sion, since TLR3 is an endosomally expressed receptor and its response requires li-
mechanism explaining multi-receptor targeted tolerance[16]. These results suggest
follow a systematic response pattern in trauma and sepsis patients. Although TLR9
-
in sepsis blood cells as compared to trauma and healthy controls. The same is true
whole blood stimulation assays[17] and may explain this difference.
Since van Endert recently described that endosomal TLR9 requires proteolytic
activation by AEP[18] it may be worth investigating whether maturation of TLR9 in
patients with sepsis/septic shock differs from the maturation status in trauma or sep-
sis. The difference between ODN2216 and ODN2006 representing type A and type
B oligonucleotides can be also explained by the recently described co-stimulatory
function of high mobility gene box-1 (HMGB-1)[19]. HMGB-1 further binds to
soluble RAGE (receptor for advanced glycation end products) and this complex is
In addition to the increased TLR9 responsiveness, sepsis patients’ blood cells
also true for ligands binding to TLR2, -3, -4, -5, and -7. This effect further supports
the concept of immune tolerance in this patients population[11].
;9(<4(:/6*205-3(44(;065(5+:,7:0:¶;:0:
danger-receptor complex [22], this molecule is in part responsive for elevations of
alone restricted to infectious complications[22]. Similarly, soluble TNF-RII binds
has been shown to be restricted to pre-treatment models[23] [24].
Healthy
1 10 100 1000 10000 100000
Pam3Csk4
FSL-1
Poly I:C
LPS
Flagellin
Loxoribin
ODN22 16
ODN20 06
no TLR-Ligand
IL-1ß concentration in pg/ml
TLR-Ligand
Healthy
1 10 100 1000 10000 100000
Pam3Csk4
FSL-1
Poly I:C
LPS
Flagellin
Loxoribin
ODN22 1 6
ODN20 0 6
no TLR-Ligand
TNF-D concentration in pg/ml
TLR-Ligand
Trauma
1 10 100 1000 10000 100000
Pam3Csk4
FSL-1
Poly I:C
LPS
Flagellin
Loxoribin
ODN22 16
ODN20 06
no TLR-Ligand
IL-1ß concentration in pg/ml
TLR-Ligand
Trauma
1 10 100 1000 10000 100000
Pam3Csk4
FSL-1
Poly I:C
LPS
Flagellin
Loxoribin
ODN2216
ODN2006
no TLR-Ligand
TNF-D concentration in pg/ml
TLR-Ligand
TLR-Ligand
Sepsis
1 10 100 1000 10000 100000
Pam3Csk4
FSL-1
Poly I:C
LPS
Flagellin
Loxoribin
ODN2216
ODN2006
no TLR-Ligand
IL-1ß concentration in pg/ml
Sepsis
1 10 100 1000 10000 100000
Pam3Csk4
FSL-1
Poly I: C
LPS
Flagellin
Loxoribin
ODN2216
ODN2006
no TLR-Ligand
TNF-D concentration in pg/ml
TLR-Ligand
Fig 3 - !"
TM
2(
232&,!$-./$0*
4HYJO [O[O4\UPJO.LYTHU`
As summarized in Table 1a, the median ratio calculated of the amount of
IL1ratio-
tion is 1.48 in healthy donors and declines to a median value of 0.18 in trauma
TNFratio, is 2.37 in healthy donors,
0.43 in trauma patients and 0.06 in sepsis patients. We conclude that these ratios
also calculated the respective IL-1ratio and TNFratio following other TLR-spe-
(data not shown).
Table 1b shows an increased release of sTNF-RII and IL-1RA following TLR4
secretion is lower in SIRS and even less in sepsis cultures. Table 2 clearly shows
their antagonists (IL-1RA, sTNF-RII)between healthy volunteers and SIRS/Sep-
sis patients. However, the amount of IL-1RA is not remarkably different between
<0.01).
Cytokine/antagonist
ratio
Healthy Trauma Sepsis
median (range) median (range) median (range)
IL-1ȕ/IL-1RA
IL-1ratio*)
1.48
(0.1-1.5)
0.18
(0.002-0.54)
0.09
(0.07-0.19)
TNF-a/sTNF-RII
TNFratio*)
2.37
(1.15-3.54)
0.43
(0.03-0.86)
0.06
(0.01-0.28)
Table 1b
Cytokine
Healthy
Trauma Sepsis
median (range) median (range) median (range)
IL-1ȕ>QJPO@ 25.5 11.2-53.8 5.2 0.0-15.3 2.8 0.3-5.0
IL-1RA [ng/ml] 11.4 8.8-18.3 23.0 8.9-39.5 26.1 4.2-54.6
TNF-Į [ng/ml] 12.2 6.0-17.8 4.54 0.2-8.4 1.9 0.3-2.7
sTNF-RII [ng/ml] 5.2 2.1-7.5 11.0 7.0-17.0 30.0 6.8-45.0
Tab. 1 a and 1 b - +,!$-,!$"1./$0./$",,!"%
TM
*
!"%*
Tab. 1 a
Tab. 1 b
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heterogeneity in the sepsis group, including cases with severe sepsis as well as sep-
tic shock. A larger study may clarify this issue.
The biomarker results presented in this pilot study supports a number of gene
-
er, are laborious expensive and time consuminghand and, considerably differ in
the expressed gene signature due to underlying diseases [25-27] and experimental
models[27]. The proteomic approach using TruCulture
TM
-
stimulation is suitable for patients undergoing different stages of a disease as well as
different diseases leading to the manifestation of severe SIRS and sepsis. The em-
well as oxidative stress by PAMP as major effectors in SIRS and sepsis.
9LMLYLUJLZ
1. Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003:348(2):138-
150.
2. Nguyen A, Yaffe MB. Proteomics and systems biology approaches to signal transduction in sepsis.
Crit Care Med 2003:31(1 Suppl):S1-6.
3. Punyadeera C, Schneider E, Schaffer D, et al. A biomarker panel to discriminate between systemic
5. Kayal S, Jais JP, Aguini N, et al. Elevated circulating E-selectin, intercellular adhesion molecule 1,
and von Willebrand factor in patients with severe infection. Am J Respir Crit Care Med 1998:157(3
Pt 1):776-784.
6. Brown KL, Cosseau C, Gardy JL, et al. Complexities of targeting innate immunity to treat infec-
tion. Trends Immunol 2007:28(6):260-266.
7. Skinner NA, MacIsaac CM, Hamilton JA, et al. Regulation of Toll-like receptor (TLR)2 and
TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens. Clin Exp Immunol
2005:141(2):270-278.
TLR-Ligand
stimulation
[LPS]
Healthy vs SIRS
Healthy vs.
Sepsis
SIRS vs. Sepsis
p-value
p-value
p-value
TNF-Į 0.012 0.0025 0.142
sTNF-RII 0.002 0.0101 0.055
IL-1ȕ 0.004 0.0025 0.211
IL-1RA 0.060 0.2677 0.760
IL1ratio
0,003 0,006 0,091
TNFratio
0,001 0,003 0,042
Tab. 2 - +45$) ./$0./$",,,!$-,!$"1
,!$./*
4HYJO [O[O4\UPJO.LYTHU`
8. Ida JA, Shrestha N, Desai S, et al. A whole blood assay to assess peripheral blood dendritic cell
function in response to Toll-like receptor stimulation. J Immunol Methods 2006:310(1-2):86-99.
9. Weiss M, Huber-Lang M, Taenzer M, et al. Different patient case mix by applying the 2003 SCCM/
surgical patients: a retrospective observational study. BMC medical informatics and decision mak-
ing 2009:9:25.
10. Bone RC. Sir Isaac Newton, sepsis, SIRS, and CARS. Crit Care Med 1996:24(7):1125-1128.
11. Hotchkiss RS, Coopersmith CM, McDunn JE, et al. The sepsis seesaw: tilting toward immunosup-
pression. Nat Med 2009:15(5):496-497.
12. Oberholzer A, Oberholzer C, Moldawer LL. Sepsis syndromes: understanding the role of innate and
acquired immunity. Shock 2001:16(2):83-96.
13. Jacinto R, Hartung T, McCall C, et al. Lipopolysaccharide- and lipoteichoic acid-induced tol-
erance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase. J Immunol
2002:168(12):6136-6141.
14. Sabroe I, Jones EC, Usher LR, et al. Toll-like receptor (TLR)2 and TLR4 in human peripheral blood
granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. J Immunol
2002:168(9):4701-4710.
15. Matsuguchi T, Musikacharoen T, Ogawa T, et al. Gene expressions of Toll-like receptor 2, but not
-
munol 2000:165(10):5767-5772.
16. Lehner MD, Morath S, Michelsen KS, et al. Induction of cross-tolerance by lipopolysaccharide and
-
tors. J Immunol 2001:166(8):5161-5167.
17. Mangsbo SM, Sanchez J, Anger K, et al. Complement activation by CpG in a human whole blood
loop system: mechanisms and immunomodulatory effects. J Immunol 2009:183(10):6724-6732.
18. van Endert P. Toll-like receptor 9: AEP takes control. Immunity 2009:31(5):696-698.
19. Tian J, Avalos AM, Mao SY, et al. Toll-like receptor 9-dependent activation by DNA-containing
immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 2007:8(5):487-496.
20. Krieg AM. TLR9 and DNA ‘feel’ RAGE. Nat Immunol 2007:8(5):475-477.
21. Barksby HE, Lea SR, Preshaw PM, et al. The expanding family of interleukin-1 cytokines and their
-
tion. Cell Death Differ 2007:14(1):10-22.
23. Guo Z, Wang S, Jiao Q, et al. Soluble TNFR II/IgG1 Fc fusion protein treatment in the LPS-me-
2009:63(7):537-542.
24. Fisher CJ, Jr., Agosti JM, Opal SM, et al. Treatment of septic shock with the tumor necrosis fac-
tor receptor:Fc fusion protein. The Soluble TNF Receptor Sepsis Study Group. N Engl J Med
1996:334(26):1697-1702.
25. Knight PR, Sreekumar A, Siddiqui J, et al. Development of a sensitive microarray immunoas-
say and comparison with standard enzyme-linked immunoassay for cytokine analysis. Shock
2004:21(1):26-30.
sterile SIRS and early sepsis. Annals of surgery 2007:245(4):611-621.
27. Chung TP, Laramie JM, Meyer DJ, et al. Molecular diagnostics in sepsis: from bedside to bench.
Journal of the American College of Surgeons 2006:203(5):585-598.
