Sepsis Inflammation Cytokines

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SepsisInflammationCytokines
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Dara Diviney Eilis Mc Govern Ross Mc Manus Edel
Duggan Michael O Dwyer Emma Caraher Brian O
Connell Patrick Stordeur Kathy Gately Ronan
Ryan Anthony Ryan Mary White Jacintha
Thornton Arun Mankan Felicity Dempsey Vivion
Crowley Dermot Kelleher
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Sepsis v SIRS

• Need a human model
• Post cardiac surgery vasodilation a good model
  for SIRS
• Sepsis compare Intensive care patients with
  sepsis to controls and to bacteraemic ward
  patients.
• New Technology RT-PCR - mRNA

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Study-SIRS

• Use RT-PCR to examine changes in TNFa, IL-10,
  mRNA from PBMC after cardiac surgery.
• Baseline and samples 1 and 6 hours after
  cardiopulmonary bypass.
• Compare subsequent samples to base line
• Compare patients requiring vasopressor support to
  those who do not require support.
• Choose elective surgical patients.
• Study immediate post operative hours.

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Patient Demographics and Operative
Details No Vasopressor Vasopressor p
value Number 48 34 Age (years) 62.5 ?
1.5 64.3 ? 1.5 NS Sex (MF) 408 2410 NS Bo
dy Surface Area (m2) 1.91 ? 0.03 1.90 ?
0.03 NS Weight (Kg) 80.5 ? 1.9 79.5 ?
2.5 NS Hypertension 20 17 NS Myocardial
Infarction 19 8 NS Diabetes Mellitus 6 7 NS
CABG 35 22 NS Valve 11 9 NS CABG/Valve 2
3 NS CPB time (min) 95.5 ? 5.1 111.5 ?
4.9 0.034 Cross Clamp time (min) 55.8 ?
3.4 71.8 ? 4.4 0.0049 __________________________
_______________________________________ Values
quoted as mean ? standard error for continuous
variables.
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Multivariate Analysis

• Parameter
• Chi Square p
• Cardiopulmonary Bypass Time ( Min) 7.26 0.007
• IL-10 TNF ratio ( 1 hr) 8.8 0.003
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• R2 0.195 N 75

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HAPLOTYPE ANALYSIS FOR TNF 82 patients
Uncomplicated ( U ) vs Complicated ( C )
Description G?A G?A C?A C?T
G?A 308 252 863 857
238 Pop Freq p value
 GACCG U 0.33 0.63 C 0.33 AGCCG U 0.28 0.
17 C 0.18 GAACG U 0.15 0.53 C 0.15 GGCC
G U 0.11 0.08 C 0.23 GACCA U 0.1 0.3 C
0.05 GACTG U 0.03 0.2 C 0.06
 
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HAPLOTYPE ANALYSIS for IL 10 82 patients
Uncomplicated ( U ) vs Complicated ( C )
Haplotype 1082 819 592 G?A C?T C?A
pop freq p value  GCC Stable 0.61
Vasopressor 0.45 0.15ACC Stable 0.22 Vasopr
essor 0.18 0.66ATA Stable 0.17 Vasopressor
0.37 0.04
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Nosocomial Respiratory Infection

• Relation between change in IL-10 TNF ratio 1
  hour after cardiopulmonary bypass and subsequent
  infection
• N Median (25 -75)
• Yes 9 17.7 4.3 - 44.6
• No 68 4.3 1.6 -11.3
• Wilcoxon Rank Sum test
• N 77, P 0.014

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Impression

• TNF a not related to haemodynamic instability in
  this model of SIRS
• IL-10 may be protective acutely, but predispose
  to subsequent infection.
• Are there any other genes associated with
  vasodilation after cardiac surgery?

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Sepsis

• Use RT-PCR to examine cytokine gene expression in
  patients with sepsis.
• Is there a reliable marker of outcome?
• Can provide absolute quantification of mRNA in
  copy numbers.
• Use PBMC as these cells are likely key in immune
  response to infection.

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Design

• Serial testing in patients with severe sepsis.
• Control group
• Bacteraemic group, without severe sepsis.
• TNFa, IL-10, IL-12, Interferon gamma.
• Express mRNA as copy numbers per 106 copies of a
  house keeping gene b actin.

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Demographics Septic Shock Sepsis Infection
p value N 42 20 10 Male 26 (61) 10
(50) 4 (40) nsa Age 70 (56-77) 62
(43-72) 82 (62-85) 0.017 SAPS2 score 47
(39-59) 35 (28-41) 27 (21-33) lt0.0001 SOFA
score 9 (8-12) 4 (3-5) 0.5 (0-1)
lt0.0001 Duration of 9 (3-21) 2 (0-17)
0.049 ventilation Duration ICU 11 (4-25) 5
(2-22) ns Site of infection Respirator
y 21 (50) 11 (55) 4 (40)
ns Abdominal 14 (33) 7 (35) 3 (30)
ns Other 8 (19) 2 (10) 3 (30) ns
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The occurrence of sepsis by category of IL-10 and
Interferon gamma mRNA.
r2 0.5, p 0.0001
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Sepsis Day 1

• Day 1 - no cytokine gene expression correlated
  with the occurrence of shock.
• Day 1 - compare upper quartile Interferong gene
  expression with remainder
• 0/13 deaths v 13/39 deaths, p 0.02.

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Sepsis Day 7

• What is pattern of cytokine mRNA after 7 days of
  critical care?
• Does this pattern predict outcome?
• Relevant outcomes are Shock and Mortality

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• Relation between categories of cytokine mRNA and
  persistent shock in patients with sepsis.
• TNF mRNA High TNF mRNA Low
• INF mRNA High INF mRNA Low
•  N 30 19 p
• Shock 1 (3) 14 (74) 0.0001
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• Death 3 (10) 5 (26) ns
• SOFA Score 3 (0-6) 10 (7-14) lt0.001
• IL-6 ( By Elisa in pg/ml)
• N 21 15
• IL-6 10.7 (0-103) 142 (29 456) 0.007

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IL-23, IL27, IL-18

• Apply to same model of infection.
• IL-23 is heterodimer p40 in common with IL-12
  and a p19
• Produced by DC, Monocytes, NK cells.
• Acts on macrphages PMN to increase Inflammatory
  cytokine production (TNFa)
• Acts on CD4 Th17 cells to increase production of
  IL-17

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IL-23, IL27, IL-18

• IL-27 is a heterodimer
• Consists of IL-27 p28 and EBV inducer gene 3
• Acts on CD4 Th 1 cells to inhibit IL-17
  production.
• IL-18 putative inducer of Interferon gamma
  production

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Relation with TNFa gene mRNA levels
On the first day of critical illness there was no
relationship between TNFa mRNA levels and the
levels of IL-18, IL-23 or IL-27 mRNA. On day 7
of critical illness there was a significant
correlation between IL-23 mRNA levels and TNFa
mRNA levels (n49, Spearmans Rho
0.56,plt0.0001).
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Vasodilation after Cardiac Surgery

• Genotype a cohort of elective cardiac surgical
  patients.
• Examine linkage with other SNP on chromosome 6.
• Categorise patients according to requirement for
  vasopressors immediately after surgery

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Relation between requirement for vasopressor
infusion and haemodynamics.   Group Vasopressor
No Vasopressor n 56 n 180 MAP 0-3
Hours(1) 74/-1.3 80/-0.7 MAP 3-6 Hours
(1) 71/-1.2 78/-0.7 MAP 6-9
Hours(1) 71/-1.2 77/-0.7 MAP 9-12
Hours(1) 72/-1.2 74/-0.7 Heart Rate (2)
87/-0.7 82/- 1.2
CVP(3) 13 /-0.4 12 /-0.2
n 22 n
13 Cardiac Index (L/M2/Min) 3 /-0.1
2.7 /-0.2 SVRI ( dy/cm/sec2)(4)
1591/-95 2130/-124   Values are
means /- SEM. ANOVA for repeated measures for
difference between groups in MAP at 3,6,9and 12
hours, p 0.0001.(2)   P 0.0007 (3)   P
0.006 (4)   P0.002
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Allele frequency for specific polymorphic alleles
in relation to requirement for vasopressor
infusion after cardiac surgery.
Gene Allele Frequency Frequency p No
Vasopressor Vasopressor TNFa -857 (C/T) 0.944 /
0.055 0.954 / 0.045 ns -863 (C/A) 0.880 /
0.119 0.845 / 0.154 ns LTA 252
(A/G) 0.611 / 0.388 0.491 / 0.509
0.03 CSNK2B 2054 (A/G) 0.828 / 0.172 0.855
/ 0.145 ns 4151 (A/G) 0.211 /
0.789 0.218 / 0.781 ns IkBL -62 (A/T) 0.620 /
0.380 0.527 / 0.472 ns DDAH II -449
(G/C) 0.702 / 0.297 0.590/0.409 0.03
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HomozygousDDAH II 449 G allele.16 patients in
the vasopressor group (28.6) homozygous for the
DDAH II G allele. 91 patients (50.5) in the
group without vasopressor infusion homozygous
for the DDAH II G allele. (OR 0.4, 95 CI
0.21-0.76 p 0.005)
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Multivariate Logistic Regression Model of the
Factors Associated with Vasopressor infusion
after Cardiac Surgery.   Factor p Odds
Ratio CI. DDAH II GG 0.008 0.4 0.2
0.77Prior Myocardial Infarct 0.0002 2.7 1.4
5.1 CPB gt 115 Minutes 0.02 2.3 1.16 4.59   N
235, p 0.001, R2 0.1   DDAH II GG versus
GC and CC CPB , cardiopulmonary bypass time.
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Frequencies of two common haplotypes amongst the
vasopressor positive and negative groups
  DDAH, LTA, IKBL, TNF-863,TNF-857
p-value
p-value Frequencies



(F-test)
(permutation) No Pressor Vasopressor
1 2
2 2 2 0.017
0.01 0.364
0.224 2 1 1
2 2 0.03
0.02 0.182
0.293     For each individual allele 1
represents the common allele and 2 represents the
rarer allele. For DDAH II 1 G, for LTA 1 A, for
IKBL A1, for TNFa 863 C1 and for TNFa 857
C1.
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Vasodilation after Cardiac Surgery

• Linked with a identifiable haplotype
• ? Related to DDAH II gene expression
• ? Related to gene expression in an adjacent gene
• Haplotype spans MHC region
• ? Related to adjacent vasoregulatory genes such
  as Endothelin 1

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Conclusions

• Distinct patterns of cytokine mRNA linked with
  outcome.
• Greater Pro inflammatory Cytokine mRNA levels are
  linked with better outcome.
• So what mediates Shock In Sepsis ?