Interleukin-6 protein expression is more important than interleukin-6 mRNA levels in assessing surgical invasiveness.
ABSTRACT Interleukin-6 (IL-6) protein has been recognized as a sensitive marker of surgical stress response. However, little is known about the clinical significance of IL-6 mRNA levels as a marker of surgical stress. This study aims to examine the role of IL-6 mRNA expression in comparing the tissue invasiveness of microendoscopic discectomy (MED) and open discectomy (OD).
Twenty-three consecutive patients were randomly selected to undergo either MED or OD. The total RNA was extracted from the peripheral whole blood of patients at pre-op and at 1, 2, 4, 8, 12 h post-op. The real-time reverse transcription polymerase chain reaction (RT-PCR) using the SYBR Green I fluorescence dye and the 2(-DeltaDeltaCt) method was adopted to measure the IL-6 gene expression.
The quantitative changes of IL-6 mRNA expression in MED and OD patients at different times post-op differed significantly, P = 0.04. Experimental results indicate that the changes in IL-6 mRNA expression in OD and MED groups varied significantly at 1 h, 12 h post-op, 10.26-fold versus 4.42-fold and 52.15-fold versus 26.78-fold increase, respectively. Although IL-6 mRNA expression demonstrated an earlier difference than protein levels at 1 h post-op, IL-6 mRNA levels were found to be significantly affected after surgical procedures. Furthermore, compared with our enzyme-linked immunosorbent assay data, no significant correlation existed between IL-6 mRNA and protein levels at any post-op time interval.
We conclude that IL-6 mRNA expression using RT-PCR to extract the total RNA from a patient's peripheral whole blood is more sensitive than protein levels but can be significantly affected by surgical procedures. The enzyme-linked immunosorbent assay data on IL-6 protein expression are more consistent and significant than IL-6 mRNA levels in comparing tissue invasiveness between MED and OD.
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ABSTRACT: The magnitude of the tissue damage from surgery impacts the trauma response. This response is proportional to the severity of surgical stress. Systemic cytokines are recognized as markers of postoperative tissue trauma. Microendoscopic discectomy (MED) recently has become popular for treating lumbar disc herniations, and is associated with favorable clinical outcomes compared with open discectomy (OD). This study postulates that MED is a less traumatic procedure, and therefore has a lower surgical stress response compared to OD. In this study, a quantitative comparison of the overall effects of surgical trauma resulting from MED and OD was performed through analyzing patient systemic cytokines response. From April, 2002 to June, 2003, 22 consecutive patients who had symptomatic lumbar disc herniations were prospectively randomized to undergo either intracanalicular MED (N=10) or OD (N=12). In this study, the Vertebroscope System (Zeppelin, Pullach, Germany) was used to perform the endoscopic discectomy procedure in all MED patients. Serum levels of tumor necrosis factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) were measured before surgery and at 1, 2, 4, 8 and 24h after surgery using an enzyme-linked immunosorbent assay. Serum C-reactive protein (CRP) was measured at the same time interval. The results showed the MED patients had shorter postoperative hospital stay (mean, 3.57+/-0.98 vs. 5.92+/-2.39 days, p=0.025) and less intraoperative blood loss (mean, 87.5+/-69.4 vs. 190+/-115 ml, p=0.042). The operating length, including the set-up time, was longer in the MED group (mean, 109+/-35.9 vs. 72.1+/-17.8 min, p=0.01). The mean size of skin incision made for the MED patients was 1.86+/-0.13 cm (range 1.7-2.0 cm); and 6.3+/-0.98 cm for the OD patients (range 5.5-8 cm), p=0.001. The patients' pain severity of the involved limbs on 10-point Visual Analog Scale before operation in MED group was 7.5+/-0.3 (range 6-9) and 8+/-0.2 (range 7-9) in OD group, p=0.17; and after surgery, 1.5+/-0.2 (range 1-2) in MED group and 1.4+/-0.1 (range 1-3) in OD group, p=0.91. CRP levels peaked at 24h in both groups, and OD patients displayed a significantly greater postoperative rise in serum CRP (mean, 27.78+/-15.02 vs. 13.84+/-6.25mg/l, p=0.026). Concentrations of TNF-alpha, IL-1beta, and IL-8 were detected only sporadically. Serum IL-6 increased less significantly following MED than after OD. In the MED group, IL-6 level peaked 8h after surgery, with the response statistically less than in the open group (mean, 6.27+/-5.96 vs. 17.18+/-11.60 pg/ml, p=0.025). A statistically significant correlation was identified between IL-6 and CRP values (r=0.79). Using the modified MacNab criteria, the clinical outcomes were 90% satisfactory (9/10) in MED patients and 91.6% satisfactory (11/12) in OD patients at a mean 18.9 months (range 10-25) follow-up. Based on the current data, surgical trauma, as reflected by systemic IL-6 and CRP response, was significantly less following MED than following OD. The difference in the systemic cytokine response may support that the MED procedure is less traumatic. Moreover, our MED patients had achieved satisfactory clinical outcomes as the OD patients at a mean 18.9 months follow-up after surgery.Journal of Orthopaedic Research 04/2005; 23(2):406-11. · 2.88 Impact Factor
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ABSTRACT: The analysis of cytokine profiles plays a central part in the characterization of disease-related inflammatory pathways and the identification of functional properties of immune cell subpopulations. Because tissue biopsy samples are too small to allow the detection of cytokine protein, the detection of mRNA by RT-PCR analysis is often used to investigate the cytokine milieu in inflammatory lesions. RT-PCR itself is a qualitative method, indicating the presence or absence of specific transcripts. With the use of internal or external standards it may also serve as a quantitative method. The most widely accepted method is quantitative competitive RT-PCR, based on internal shortened standards. Recently, online real-time PCR has been introduced (LightCycler®), which allows quantitation in less than 30 min. Here, we have tested its use for the analysis of cytokine gene expression in different experimental in vitro and ex vivo settings. First, we compared quantitative competitive RT-PCR with real-time RT-PCR in the quantitation of transcription levels of the CD4+ cell-specific chemoattractant Interleukin-16 during the maturation of monocyte-derived dendritic cells, and found a good correlation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoarthritis as assessed by real-time RT-PCR paralleled differences in the level of IL-16 protein in the synovial fluid. Finally, we employed real-time RT-PCR to study the cutaneous expression of several cytokines during experimental immunomodulatory therapy of psoriasis by Interleukin-10, and demonstrate that the technique is suitable for pharmacogenomic monitoring. In summary, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression even with small quantities of tissue. The results obtained do not differ from those generated by quantitative competitive RT-PCR.Journal of Immunological Methods 01/2001; · 2.23 Impact Factor
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ABSTRACT: Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.Twin Research 01/2005; 7(6):564-70.