This study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in the IL-6 and TNF-α genes and serum levels of MIP-1α and IL-6 in burn patients who developed sepsis. Blood samples were collected from 40 burn patients with sepsis and 30 burn patients without sepsis. Gene polymorphisms were detected using PCR, and serum cytokine levels were measured using ELISA. The results showed higher serum IL-6 and MIP-1α levels in patients with sepsis compared to controls. No significant differences were found in IL-6 or TNF-α allele frequencies between groups. A correlation was found between IL-6 serum levels and IL-6 genotype in patients with sepsis but not controls.

1. ‫بسم‬
‫هللا‬
‫الرحيم‬ ‫الرحمن‬
َ‫ل‬ْ‫ع‬َ‫ت‬ ‫ُن‬‫ك‬َ‫ت‬ ْ‫م‬َ‫ل‬ ‫ا‬َ‫م‬ َ‫ك‬َ‫م‬َّ‫ل‬َ‫ع‬َ‫و‬
ُ‫م‬
ۚ
ُ‫ل‬ْ‫ض‬َ‫ف‬ َ‫ان‬َ‫ك‬َ‫و‬
‫ه‬ َّ
‫اَلل‬
‫ا‬ً‫م‬‫ي‬‫ه‬‫َظ‬‫ع‬ َ‫ك‬ْ‫ي‬َ‫ل‬َ‫ع‬
﴿
113
﴾
‫صدق‬
‫هللا‬
‫العظيم‬ ‫العلي‬
(
‫سورة‬
‫النساء‬
/
‫اية‬
113
)

2. by
Raghda S. M. alomari
Supervised by
Prof.
Dr. Mahdi H. Al-Ammar

4. Introduction
Burn injury remains an important cause of
morbidity and mortality worldwide . Infection
complications , including sepsis , septic shock
and sepsis –related organ failure , are common
among patients with moderate to severe burn
injuries .
Sepsis is a life-threatening organ-dysfunction
condition caused by a dysregulated response to
an infectious condition that can cause
complications in patients with major trauma .

5. Introduction
Burns are one of the most destructive forms of
trauma; despite the improvements in medical
care, infections remain an important cause of
burn injury-related mortality and morbidity , and
complicated sepsis predisposes patients to
diverse complications such as organ failure,
lengthening of hospital stays, and increased costs
Accurate diagnosis and early treatment of sepsis
may have a beneficial impact on clinical
outcome of burn – injured patients

6. Introduction
 In sepsis , the immune response that is initiated
by an invading pathogen fails to return to
homeostasis, thus culminating in a pathological
syndrome that is characterized by sustained
excessive inflammation and immune suppression
 The increase of cytokine production both in
humans and in experimental models seems to
play important roles in the pathophysiology of
sepsis and septic shock after burn injury.

7. Introduction
Pro-inflammatory interleukin - 6 is increased
early after burn and secreted by monocytes ,
endothelial cells and fibroblasts , and is able to
stimulate B and T lymphocytes and induce fever
. Some studies have indicated that IL-6 may play
a key role in the inflammatory response to
microbial invasion and also , it is one of the most
important members which may be associated
with sepsis risk and outcome.

8. Introduction
Macrophage inflammatory protein-1 alpha (MIP-
1α) is a chemotactic chemokine secreted by
macrophages. It performs various biological
functions, such as recruiting inflammatory cells,
wound healing, inhibition of stem cells, and
maintaining effector immune response
Single nucleotide polymorphisms (SNPs) , variations in
a nucleotide at a specific chromosome location, have
been linked to sepsis susceptibility and differences in
prognosis .

9. Introduction
Among these diverse cytokines , IL-6 and tumor
necrosis factor-α (TNF-α) have attracted
considerable attention . The human IL-6 gene was
mapped to chromosome 7p21 region and
contained several single nucleotide
polymorphisms .
The G to C polymorphism at position-174 of the
IL-6 gene (rs1800795) is associated with an
adverse outcome in a number of inflammatory
diseases, but its association with sepsis remains
unclear.

10. Introduction
On the other hand , the promoter of single nucleotide
polymorphisms (SNP) at position - 308 (G to A
substitution) of TNF-α gene has been demonstrated to
play a major role in the pathogenesis of sepsis and its
complications after burn injury .
The identified specific Toll-like receptor 4 and TNF-α
SNPs associated with increased risk of sepsis after burn
injury .
TNF-α is pro-inflammatory cytokine produced by
lymphocytes and macrophage.

11. Aims of the Study
 The current study aimed to investigate the evaluate the correlation of
Single Nucleotide Polymorphisms (SNP) in IL-6 -174 G→C , TNF–α ( -
308 G →A , with serum level of MIP-1 expression in the development of
burn patients with sepsis . The following objectives :-
1.Blood sample collection in a study based on peripheral blood sample .
2.Detection of gene polymorphism in (IL-6 -174 G→C) and TNF–α (-308
G →A) using ARMS-PCR.
3. Screening for IL-6 and MIP-1 level by using ELISA test .
4.The association between gene polymorphism and cytokine levels with the
development of burn patients with sepsis .

13. Sample collection from burn
patients
Burn patient with
sepsis
5 ml of Blood sample
Serum :- ELISA test for
MIP-1α & IL-6
Whole blood :-Gene
polymorphism for IL-6 &
TNF-α
Data analysis
The diagnosis of sepsis by
PCT ,WBC &
Temperature body
Burn patient without
sepsis
Study design

15. Diagnostic of Sepsis in Burn Patients
Biomarker Sepsis n=40 Without sepsis n=30 P-value
Procalcitonin (ng/ml) 9.16±5.07 0.57±1.27 0.01*
WBC cells (1*103/cc) 12.87±5.88 7.30±3.46 0.038*
Temperature 38.43±2.43 36.82±0.19 0.47
Blood culture Positive Negative
Table 1 :- Show the result biomarkers levels in sepsis and without sepsis burn
patients

16. Table 2 :- Shows higher frequency of sepsis in burn patient at age from 24-34 year (47.5%) , there
is no significant difference in mean age between burn with sepsis and burn without sepsis
Chi-Square
Sig.
Control
(burn without sepsis)
Patient
(burn with sepsis)
Age groups
4.618
0.099 NS
13 (43.3%)
8 (20%)
15-24 year
9 (30%)
19 (47.5%)
24-34 year
8 (26.7%)
13 (32.5%)
>35 year
0.051 NS
27.53± 9.03
32.1± 9.62
Mean ± SD
15-50
18-50
Range

17. Table 3 :- Shows Gender difference between burn with sepsis and burn without sepsis groups ,
sepsis in burn was found to be more prevalent among females(60%) than males (40%)
Chi-Square
p-value.
Control
(burn without sepsis)
Patient
(burn with sepsis)
Gender
0.000
1.000 NS
18 (60%)
24 (60%)
Female
12 (40%)
16 (40%)
Male

18. Immunological Study
Study
groups
Patients
(burn with sepsis)
n=40
Control
(burn without sepsis)
n=30
healthy
Mean
(SD)
Median
(IQR)
Range
Mean
(SD)
Median
(IQR)
Range
p-
value
Mean
(SD)
Median
(IQR)
Range
IL-6
(pg/ml)
443.77
(42.63)
471.62 137.9
299
(120.11)
295.3 360.87 <0.001
60.3
(32.8)
55.5 130.4
MIP -α
(pg/ml)
148.43
(34.63)
141.9 136.12
140.4
(36.93)
144.49 124.73 0.354
15.3
(2.52)
14.22 7.94
Table 3 :- Shows the serum level and median (IQR) of IL-6 and MIP-1α were significantly higher in
patient group than in control group they were 443.77 pg/ml (471.62) versus 299 pg/ml (295.3) and
148.43 pg/ml (141.9) versus 140.4 pg/ml (144.49) respectively

19. Immunological Study
Figure 1 :- Histogram Showing the Serum Level of IL-6 in Patient and control

20. Immunological Study
Figure 2 :- Histogram Showing the Serum Level of MIP-1α in Patient and control

21. Immunological Study
Figure 3 :- Histogram Showing the Serum Level of IL-6 in Patient and control
according

22. Immunological Study
Figure4:- Histogram Showing the Serum Level of MIP-1α in Patient and control
according

23. Molecular study
Figure 5 :- Agarose gel electrophoresis image that show the T . ARMS-PCR product analysis of IL-6 in patient
sample . T . ARMS-PCR product was analysis by 2% agarose gel . Where M: marker (100bp – 1500bp ) , lane
GG wild type homozygote , lane CC mutant type homozygote, and lane GC heterozygote
Detection of interleukin-6 by gel electrophoresis

24. Molecular study
Figure 6 :- Agarose gel electrophoresis image that show the T . ARMS-PCR product analysis of IL-6 in control
sample . T . ARMS-PCR product was analysis by 2% agarose gel . Where M: marker (100bp – 1500bp ) , lane
GG wild type homozygote , lane CC mutant type homozygote, and lane GC heterozygote
Detection of interleukin-6 by gel electrophoresis

25. Molecular study
Table 4 :- IL-6 genotype frequency distribution in burn patients with sepsis and control ( burn without sepsis )
groups. The genotypes relative frequency in burn patients with sepsis were as follow : GG ( 50 % ) , GC ( 35%)
and CC ( 15%) ; while in control ( burn without sepsis ) subjects : GG ( 66.7%) , GC (26.7%) and CC ( 6.7%).
Frequency distribution of patients and control subjects according to IL-6
genotypes
IL-6 SNP
Study groups
Total P-value B OR
95% CI for OR
Patient
(burn with sepsis)
N=40
Control
(burn without sepsis)
N=30
Lower
Bound
Upper
Bound
CC
6
15.0%
2
6.7%
8
11.4%
0.210 1.099 3.000 0.539 16.689
GC
14
35.0%
8
26.7%
22
31.4%
0.304 0.560 1.750 0.602 5.087
GG
20
50.0%
20
66.7%
40
57.1%
Reference Category
P-value 0.074 0.058

26. Molecular study
Table 5 :- G allele is in higher frequency ( 67.5%) in burn patients with sepsis than C allele (32.5%) .There was
no significant differences in the IL-6 allele frequency distribution between control and patients groups (P =
0.103),
Frequency distribution of patients and control subjects according to IL-6
genotypes
IL-6
study groups
Total P-value OR
95% CI for OR
Patients
(burn with
sepsis)
Control
(burn without
sepsis)
C
26
32.5%
12
20.0%
38
27.1%
0.103 1.926 0.877 4.230
G
54
67.5%
48
80.0%
102
72.9%
Total
80
100.0%
60
100.0%
140
100.0%
P-value 0.002 0.001
RR
95% CI
1.292
0.97-1.71
0.671
0.40-1.11

27. Molecular study
Figure 5 :- Agarose gel electrophoresis image that shows the ARMS-PCR product analysis of TNF–α (-308 G
→A) in patient sample . ARMS-PCR product was analyzed by 2% agarose gel . Where M: marker (100bp –
1500bp ) . GA heterozygous , positive PCR amplification 184 bp product size.
Detection of Tumor necrosis factor alpha by gel electrophoresis

28. Molecular study
Figure 5 :- Agarose gel electrophoresis image that shows the ARMS-PCR product analysis of TNF–α (-308 G
→A) in control sample . ARMS-PCR product was analyzed by 2% agarose gel . Where M: marker (100bp –
1500bp ) . AA homozygous , GG homozygous and GA heterozygous , positive PCR amplification 184 bp product
size.
Detection of Tumor necrosis factor alpha by gel electrophoresis

29. Molecular study
Table 6 :- TNF alpha genotype frequency distribution in patients and control groups. The genotypes relative
frequency in burn patient with sepsis were as follows : GG ( 40 % ) , GA ( 45%) and AA ( 15%) ; while in
control ( burn without sepsis ) subjects : GG ( 53.3%) , GA (33.3%) and AA ( 13.3%).
Frequency distribution of patients and control subjects according to
TNF-α genotypes
TNF-α SNP
Study groups
Total P-value B OR
95% CI for OR
Patient
(burn with sepsis)
N=40
Control
(burn without
sepsis)
N=30
Lower
Bound
Upper
Bound
AA
6
15.0%
4
13.3%
10
14.3%
0.582 0.405 1.500 0.355 6.347
GA
18
45.0%
10
33.3%
28
40.0%
0.267 0.588 1.800 0.637 5.083
GG
16
40.0%
16
53.3%
32
45.7%
Reference Category
P-value 0.014 0.109

30. Molecular study
Table 7 :- G allele has higher frequency ( 62.5%) in burn patients with sepsis than A allele (37.5%) . There was
no significant differences in the TNF alpha allele frequency distribution between control and patients groups (P =
1.400),
Frequency distribution of patients and control subjects according to IL-6
genotypes
TNF-α
study groups
Total P-value OR
95% CI for OR
Patients
(burn with sepsis)
Control
(burn without sepsis)
lower upper
A
30
37.5%
18
30.0%
48
34.3%
1.400 0.686 2.859 1.400
G
50
62.5%
42
70.0%
92
65.7%
Total
80
100.0%
60
100.0%
140
100.0%
P-value 0.025 0.002
RR
95% CI
1.150
0.86-1.53
0.821
0.53-1.26

31. Table 8 :- Shows Correlation between IL-6 serum and IL-6 Genotype in Burn
Patients with Sepsis and Control Burn Patient without Sepsis
IL-6 SNP
IL-6 (pg/ml)
p-value
Patient (burn with sepsis) Control(burn without sepsis
CC
N 6 2
Mean 451.33 362.94
0.867
Std. Deviation 40.31 160.86
Median 472.07 362.94
Range 102.17 227.49
GC
N 14.00 8.00
Mean 436.67 268.53
<0.001
Std. Deviation 48.51 102.84
Median 472.07 295.30
Range 129.70 286.31
GG
N 20.00 20.00
Mean 446.46 304.80
<0.002
Std. Deviation 40.38 126.11
Median 470.80 292.17
Range 137.90 360.87

32. Table 8 :- shows the summary of correlations of serum IL-6 , serum MIP-1α and IL-6
SNP , TNF- α SNP
IL-6
(pg/ml)
MIP -α
(pg/ml)
TNF-α SNP IL-6 SNP
Patient
(burn with sepsis )
Correlation
Coefficient
Age 0.134 0.028 0.230 0.076
MIP -α (g/ml) 0.134 1.000 0.114 0.112
TNF-α SNP 0.122 0.114 1.000 0.234
IL-6 SNP 0.011 0.112 0.234 1.000
Sig. (2-tailed) Age 0.410 0.862 0.153 0.642
MIP -α (g/ml) 0.409 . 0.484 0.491
TNF-α SNP 0.454 0.484 . 0.146
IL-6 SNP 0.948 0.491 0.146 .
Control
(burn without sepsis )
Correlation
Coefficient
Age 0.167 0.098 0.547* 0.090
MIP -α (g/ml) 0.172 1.000 0.396* 0.153
TNF-α SNP 0.008 0.396* 1.000 0.006
IL-6 SNP 0.057 0.153 0.006 1.000
Sig. (2-tailed) Age 0.377 0.607 0.002 0.638
MIP -α (g/ml) 0.362 . 0.030 0.419
TNF-α SNP 0.967 0.030 . 0.975
IL-6 SNP 0.766 0.419 0.975 .

34. Conclusions
1.Sepsis is a medical emergency commonly encountered
during the treatment of burn patients
2.PCT assay can be a valuable complement to the clinical
diagnosis of sepsis and is promising as a method to reduce
antibiotic resistance in critically ill patients.
3.Higher level of serum IL-6 in burn patients with sepsis
compared with burn patients without sepsis individuals .
This plays a role in diagnostic of sepsis .

35. Conclusions
4. MIP-1α level play an important role in burn patients with
sepsis and without sepsis .
5.The presence of heterozygous GA genotype in the TNF-α-308
was associated with sepsis of burn patients .
6.The present study showed that heterozygous GC genotype was
significantly higher in correlation between IL-6 serum and IL-6
genotype in burn patients with sepsis and burn patients without
sepsis groups (P < 0.001). Also , homozygous GG genotype was
significantly higher in correlation between IL-6 serum and IL-6
genotype in burn patients with sepsis and burn patients without
sepsis groups (P < 0.002) .

36. Recommendations
1.Studying more gene polymorphism associated with burn patients with
sepsis and burn patients without sepsis .
2.Studying MIP-1α genotype in burn patients with sepsis and compared
with healthy individuals.
3.Studying correlation between TNF-α serum and TNF-α genotype in
burn patients with sepsis and without sepsis .
4.detect other cytokines effect on the development of sepsis .
5.The use of tetra ARMS-PCR to detect genotype single-nucleotide
polymorphisms (SNPs) and allows the study of SNPs in a fast, reliable,
and low-cost way.

37. Thank you
for listening