Toll-like receptor 4 (TLR4), a receptor of innate immunity, provides the site for the lipopolysaccharide shell of Gram-negative organisms to bind and initiate the inflammatory cascade in sepsis. Its expression and polymorphisms (Asp299Gly and Thr399lle) have been studied in various disease states (sepsis, asthma, atherosclerosis, immunodeficiencies), ethnic groups, ages, levels of physical training, gestational status and trauma [1–7]. The expanding body of evidence in regard to TLR signalling is demonstrating the importance between such signalling and human disease. A translational research project between the Departments of Anaesthesiology and Medical Microbiology and Immunology was initiated to determine any difference in the amount of TLR4 expression in elective preoperative patients in regard to height, weight, age, gender, body surface area, body mass index, hypertension and diabetes. Race was not analysed.
After institutional research board approval a prospective study of the blood specimens of 52 consecutive elective, preoperative patients was performed. The blood was directly decanted into a PAXgene Blood RNA tube (Qiagen) designed for the stabilization of RNA from whole blood. Whole blood RNA was extracted using the PAXgenez™ Blood RNA System (PreAnalytiX, Hombrechitikon, CH), including treatment with DNase I to prevent genomic DNA contamination (Qiagen). Extracted RNA was stored at 80°C until required for copy of DNA (cDNA) synthesis. Quantification of TLR4 expression levels in human lymphocytes was by real time polymerase chain reaction (RT-PCR) and results compared to standard curves. Pearsons's correlation coefficient was used for statistical analysis of the continuous variables and t-test was used in the analysis of the categorical variables (see Tables 1 and 2). The TLR4 concentration varied from 0.211 to 2.490 fmol μL−1 per cDNA, however there was no statistical significance between the concentration of TLR4/cDNA expressed by RT-PCR and the continuous variables: height, weight, body mass index or surface area and age, nor the categorical variables: hypertension, diabetes and gender. None of the patients had postoperative infections upon follow-up a week later.
This study was an initial attempt to quantify the TLR4 receptor expression by elective patient variable preoperatively. While we found no variation of TLR4 in our elective preoperative patients we are cognizant of several limiting factors in our pilot study: (1) the small size of our cohort introduces the possibility of a Type II error, (2) the Asp299Gly and Thr399lle TLR4 polymorphisms were not addressed and (3) race was not evaluated. However, we plan to extend our epidemiological investigation of TLR4 in the perioperative setting because it would be fascinating to determine if the expression of TLR4 and its quantitative determination are similar in all humans regardless of physical characteristics (polymorphisms included). It is noteworthy that a 10-fold variability in the concentration of TLR4/cDNA was not of statistical significance in this study thus raising an interesting scientific interrogative as to the biological significance of this 10-fold difference in regard to innate body defences if an infection were to become established. This matter remains to be investigated. Understanding how TLR4 receptors influence the human immune response and how the lipopolysaccharide of Gram-negative bacteria evades defences may allow future physicians and scientists to alter or manipulate TLR4 activation in order to provide for improved postoperative outcomes.
Additionally, placing biological results aside, this endeavour allowed bench researchers and clinicians to engage in a collaborative, translational relationship that provided an opportunity for anaesthesiologists to take their practice to the laboratory and to potentially explore the immunological basis for their actions.
T. J. Papadimos
L. Y. Chen
Z. K. Pan
1Department of Anesthesiology, Immunology and Medical Microbiology, Medical University of Ohio, Toledo, OH, USA
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