presentation of my thesis
- 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.