| Objective: The present study was performed to investigate: (1) whether the polymorphism of lipopolysaccharide binding protein (LBP Phe436Leu), CD 14 (C-159T) and Toll like receptor 4 (TLR4 Asp299Gly), TLR4 (Thr399Ile) influence the cytokine expression of whole blood to lipopolysaccharide (LPS) in vitro; (2) the potential regulatory role of LBP Phe436Leu, CD14 C-159T, TLR4 Asp299Gly and TLR4 Thr399Ile polymorphism in host inflammatory response in patients with extensive burns; and (3) the relation of LBP Phe436Leu, CD14 C-159T, TLR4 Asp299Gly and TLR4 Thr399Ile polymorphism to sepsis susceptibility and prognosis in patients following major burns.Methods: (1) Healthy blood donors aged 18 to 51 yrs were included in this study. Blood samples were collected in apyrogenic tubes containing EDTA-K2. The blood was processed immediately and diluted 1:1 (v/v) with RPMI 1640. Whole blood culture was performed and divided into two groups. One of them served as normal controls without LPS incubation, the other was experimental group stimulated with 100ng/ml LPS at 37 C with 5%CO2 for 12 hrs. Supernatant and blood cells were separately stored frozen at -70 C until further analysis. (2) 26 patients with burns covering more than 30% of body surface area aged 19 to 57 yrs were studied. These patients were divided into severe burned group and extremely severe burned group. Blood samples wereobtained from patients on days 1, 3, 7, 14, 28 postburn and collected in apyrogenic tubes containing EDTA-K2. Plasma and blood cells were separately stored frozen at -70 C until further analysis. LBP Phe436Leu, CD 14 C-159T, TLR4 Asp299Gly, and TLR4 Thr399Ile polymorphism were determined by polymerase chain reaction and subsequent restriction enzyme digestion of the polymerase chain products. LBP, CD 14, and tumor necrosis factor-a (TNF-a) mRNA expressions were semi-quantitative determined by reverse transcription polymerase chain reaction (RT-PCR), taking -actin as an internal standard. The concentrations of LBP, soluble CD14 (sCD14), TNF-a, interleukin 6 (IL-6) and IL-10 were determined by enzyme-linked immunoadsorbent assay (ELISA). Correlations between genotypes and the incidences of sepsis as well as multiple organ dysfunction syndrome (MODS) were investigated in extensive burn patients.Results: (1) LBP, CD14 and TLR4 genotyping were performed in 118 healthy blood donors. The frequency of LBP 436Phe and LBP 436Leu allele were 94.1% and 5.9%, respectively. 14 subjects were heterozygous (Phe/Leu, 11.9%), and the others were all homozygous for Phe allele (Phe/Phe, 88.1%). The frequency of CD 14-159 T and CD 14-159 C allele were 60.2% and 39.8%, respectively. 40 subjects were homozygous for T allele (TT, 33.9%), 62 were heterozygous (TC, 52.5%), and 16 individals had the genotype CC (13.6%). We did not find TLR4 Asp299Gly and TLR4 Thr399Ile polymorphism in the 118 individuals. (2) LBP 436 Phe/Leu heterozygotes tended to have higher LBP levels than 436Phe homozygotes either before or after LPS stimulation, while no marked correlations were found between LBP Phe436Leu polymorphism and TNF-a, IL-6 or IL-10 formation to LPS as well as CD14expression. (3) CD14C-159T polymorphism was associated with CD 14 mRNA expression in leukocytes and sCD14 release in supernatant. C homozygotes had the lowest CD 14 mRNA expression and sCD14 levels among the three genotypes either before or after LPS stimulation. (4) The subjects homozygous for T allele had the highest TNF- while the lowest IL-10 supernatant concentration in the three genetypes. (5) LBP, CD 14 and TLR4 genotyping were performed in 26 patients with burns covering more than 30% of body surface area. The frequency of LBP 436Phe and LBP 436Leu allele were 98.1% and 1.9%, respectively. Only one case was heterozygous (Phe/Leu, 3.8%), and the others were all homozygous for the Phe allele (Phe/Phe, 96.2%). The frequency of CD14-159T and CD14-159C allele were 57.7% and 42.3%, respectively. 8 patients were homozygous for T allele (TT, 30.8%), 14 were heterozygous (TC, 53.8%), and 4 had ge...
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