orthotopic liver transplantation ( olt ) is the most effective and the best therapeutic solution for final stage liver diseases . systemic inflammatory response syndrome(sirs ) often accompanies sepsis , trauma , hypoxia , and ischemia - reperfusion injury ( iri ) [ 24 ] . the activation of mononuclear phagocytes , and consequently release of massive amount of proinflammatory cytokines may lead to multiorgan dysfunction syndrome ( mods ) [ 3 , 4 ] . sirs is a common feature after major surgery [ 5 , 6 ] . in recent years , sirs has been interpreted as a warning sign for postoperative complications and organ failure [ 79 ] . specifically , longer sirs duration has been associated with poor outcomes after surgery [ 5 , 6 , 8 , 9 ] . toll - like receptors ( tlrs ) play an important role in many pathophysiological processes such as inflammation and iri [ 1014 ] . tlr2 and tlr4 are members of the tlrs family [ 13 , 14 ] , and could initiate inflammatory responses to various stimuli [ 2 , 15 ] . a previous study in this laboratory revealed increased expression of tlr2/4 in mononuclear and proinflammatory cytokines in liver transplantation . based on this finding , we speculate that tlr2/4 may also contribute to the development of sirs during olt . in the current study , we examined the expression of tlr2/4 on pbmc in a group of olt patients with sirs , and compared the results to that in a group of patients without sirs . this study was approved by the research ethics board of the third affiliated hospital , sun yat - sen university . twenty - seven patients ( 24 males and 3 females ) with end - stage liver diseases undergoing modified piggyback liver transplantation were enrolled . among these patients , 12 had hepatitis b cirrhosis , 8 had small liver cancer ( tumor diameter <3 centimeter ) on a hepatitis b background , 4 had chronic severe hepatitis b and the remaining 3 had drug - related acute liver failure . liver function based on modified child - pugh classification   was a in 10 patients , b in 5 patients , and c in the remaining 12 patients . physical status of the patients was iii or iv according to the american society of anesthesiologists ( asa ) classification ( table 1 ) . the status of organ donors was cardiac death in 8 cases , brain death in 10 cases , and living relatives in 9 cases . warm ischemia for donation after cardiac death was 3 to 4.5 minutes ( table 2 ) . body temperature was keep at 36~37c by a convective air warming system and fluid warming system . to avoid interference of measurements of tlr2/4 expression on mononuclear cells , only concentrated red blood cells , fresh frozen plasma and cryoprecipitate were transfused during operation according to monitoring . all patients received modified piggyback liver transplantation with venous reformation and no veno - venous bypass ( vvbp ) . surgical management of first and second hepatic hilums is similar to classic orthotopic liver transplantation but without short hepatic vein disposal . after disconnection of the first hepatic hilum , vena cava was interrupted by a satinskys clamp from the back of the liver . the openings of hepatic veins on the anterior wall of the vena cava were connected to form an open inverted triangular cuff . the posterior wall of the donor inferior vena cave ( ivc ) was incised to fashion a wide - open inverted triangular cuff that matched the ivc opening in the recipient . hepatic artery and bile duct anastomoses were completed [ 1820 ] .   the entire procedure consisted of an anhepatic phase ( from vascular clamping to reperfusion of portal vein and inferior vena cava ) and a neohepatic phase ( from reperfusion of donor liver to the end of operation ) . the demographic data as well as the child - turcotte - pugh ( ctp ) scores , asa classification , duration of the operation , volume of blood loss and input were collected . duration of postoperative mechanical ventilation , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , prothrombin time ( pt ) , blood urea nitrogen ( bun ) , and serum creatinine ( scr ) were also recorded . whole blood ( 4  ml ) was collected at t1 ( after induction of anaesthesia ) , t2 ( 25 minutes after the beginning of anhepatic phase ) , t3 ( 3 hours after graft reperfusion ) , and t4 ( 24 hours after graft reperfusion ) . two ml of blood sample was collected in edta tubes for analysis of tlr2/4 immediately with flow cytometry . the remaining two ml of blood sample was collected in dry tubes for tnf- , il-1 , and il-8 assay . twenty l allophycocyanin ( apc ) antihuman cd14 ( ebioscience ) plus 20  l fluoresceinisothiocyanate ( fitc ) antihuman toll - like receptor 2 ( ebioscience , san diego , california , usa ) or phycoerythrin ( pe ) antihuman toll - like receptor 4 ( ebioscience ) were added to 100  l of edta treated blood . after which , they were mixed with 2  ml of rbc lysis buffer ( ebioscience ) in the dark for 15 minutes and then centrifuged for 5 minutes at 300  g. after rinsing twice , the supernatant was discarded and sample was preserved at 4c in the dark . samples were quantified with facs calibur flow cytometry ( becton disckinson , franklin lakes , new jersey , usa ) . the isotype controls were fitc mouse igg2 and pe mouse igg2 ( ebioscience ) . tnf- , il-1 , and il-8 were measured with elisa ( rapidbio , west hills , california , usa ) . patients ' temperature , heart rate , respiratory rate , and white blood cell count were assessed every 6 hours for 7 days after the operation . the diagnosis of sirs was based on the presence of two or more of the following criteria , verified by an icu physician as well as an anesthesiologist : ( 1 ) temperature > 38c or < 36c , ( 2 ) heart rate > 90 per minute , ( 3 ) respiratory rate > 20 per minute or paco2 < 32  mmhg , and ( 4 ) white blood cell count > 12,000/ml , < 4,000/ml , or > 10% immature ( band ) forms . one - way anova was used to analyze the difference between the different phases in the same group . independent - samples t - test was used to analyze the difference between the sirs and non - sirs groups . data of nonnormal distribution are expressed as median ( interquartile range ) [ median ( q ) ] , and were analyzed by wilcoxon signed ranks test . twelve out of 27 patients developed sirs after olt ( at 6 to 78 hours ) , and two died of lung infection . there were no significant differences between two groups on graft origination , duration of cold or warm ischemia ( table 2 ) . blood loss and concentrated red blood cell ( rbc ) transfusion during the operation were larger in the sirs group than in the non - sirs group . the pre - anhepatic phase and anhepatic phase lasted longer in sirs patients ( table 3 ) . ctp score , age , gender , body weight , ascites , urinary production , the length of neohepatic phase or the entire operation did not differ between the sirs and non - sirs groups ( table 3 ) . duration of postoperative mechanical ventilation in the sirs group was significantly longer than non - sirs group . hepatic function , renal function , and infection ( respiratory tract ) after the surgery also did not differ between the 2 groups ( table 4 ) . the baseline tlr2 on pbmc was 74% ( interquartile range : 25% ) and 80% ( interquartile range : 28% ) in sirs and non - sirs groups ( p > .05 , figure 1 ) . baseline tlr4 was 12% ( interquartile range : 8% ) and 18% ( interquartile range : 21% ) in sirs and non - sirs groups ( p > .05 , figure 2 ) . tlr 2 expression was significantly higher at t3 and t4 in comparison to t1 and t2 in the sirs patients but not in the non - sirs group ( figure 3 ) . the expression of tlr2/4 , and particularly relative increase at t3/4 over t1 , in two died patients were higher than the average . baseline tnf- , il-1 , and il-8 was 90 ( interquartile range : 118 ) , 34(interquartile range : 239 ) , and 163(interquartile range : 181 ) pg / ml in sirs group , and 96(interquartile range : 488 ) , 38(interquartile range : 161 ) , and 64(interquartile range : 173 ) pg / ml in the non - sirs group , respectively . serum tnf- , il-1 , and il-8 was significantly higher at t3 and t4 in comparison to t1 and t2 in the sirs group , but not in the non - sirs group ( figures 5 , 6 , and 7 ) . the expression of tlr4 , but not tlr2 , was positively correlated to serum tnf- ( r = 0.310 ,  p = .029 ) , and il-8 ( r = 0.304 ,  p = .025 ) in the sirs group but not in the non - sirs group . in the sirs patients , the increase of tlr4 at t4 was positively correlated with the length of anhepatic phase ( r = 0.688 ,  p = .013 ) . the current study demonstrated that expression of tlr2/4 on pbmc and concentration of inflammatory cytokines after liver transplant reperfusion were significantly higher in patients with sirs than those without . sirs is an inflammatory state caused by serious trauma , and infection [ 24 , 21 ] . a cardinal feature of sirs is the activation of inflammatory cells such as monocyte - macrophages , neutrophils , and massive release of proinflammatory cytokines [ 2 , 4 ] . sirs is common in olt due to surgical trauma , hemorrhage , and ischemia - reperfusion injury . tlr2/4 on immune cells can activate nuclear factor kappa b ( nf-b ) and activator   protein-1 ( ap-1 ) in response to a variety of pathological conditions , which in turn initiate or amplify inflammation , and ultimately , organ injury [ 1114 ] . lipopolysaccharide ( lps ) can induce tlr4 gene expression in granulocyte and endothelial cells , and activate nf-b and the production of tnf- , il-6 , and il-8 . previous studies also demonstrated that high expression of tlr4 is positively correlated with ischemia - reperfusion injury . importantly , the transcriptional and translational signal of tlr2/4 in mononuclear cell was upregulated significantly in sirs patients . although there was no significant difference in ctp scores between the sirs and non - sirs groups , the expression of tlr2/4 on pbmc and serum proinflammatory cytokines at t3 and t4 were significantly higher in the sirs group . the increase of tnf- , il-1 or il-8 was also positively correlated with the expression of tlr4 in monocytes in the sirs group , suggesting that high expression of tlr2/4 in olt patients is associated with sirs . previous studies indicated that cytokine , endotoxin are involved in the regulation of tlr2/4 expression [ 2630 ] . within the context of liver transplantation , factors that could upregulate tlr2/4 may include prolonged surgery , massive blood loss and transfusion , liver ischemia / reperfusion , translocation of enteric microbes during portal vein occlusion and reopening , and ischemia - reperfusion injury of graft [ 3133 ] . specifically , blood loss , the length of preanhepatic phase and anhepatic phase may be the most important factors for upregulation of tlr2/4 expression in pbmc after olt .   in conclusion , our findings suggest that upregulation of tlr2/4 on pbmc could initiate sirs after major surgery such as olt .
background . to explore the relationship between toll - like rpheral blood mononuclear cells ( pbmc ) and systemic inflammatory response syndrome ( sirs ) in postoperative patients of liver transplantation ( lt ) . methods . 
 blood samples of 27 patients receiving lt were collected at t1 ( after induction of anaesthesia ) , t2 ( 25 minutes after the beginning of anhepatic phase ) , t3 ( 3 hours after graft reperfusion ) , and t4 ( 24 hours after graft reperfusion ) . 
 the expression of tlr2/4 on pbmc and serum concentration of tumor necrosis factor ( tnf)- , interleukin ( il)-1 , and il-8 were measured . 
 the patients were divided into sirs 
 group ( n = 12 ) and non - sirs group ( n = 15 ) for analysis . 
 results . blood loss and transfusion 
 were higher in the sirs group than in the non - sirs group . 
 both the preanhepatic and anhepatic phase were significantly longer in the sirs group . 
 the tlr2/4 expression on pbmc as well as serum tnf- , 
 il-1 , and il-8 were significantly 
 higher at t3 and t4 than that at t1 and t2 in the sirs patients . 
 the expression of tlr4 on pbmc is positively correlated to serum tnf- , il-8 . 
 expression of tlr2/4 on pbmc and serum 
 concentrations of tnf- , 
 il-1 , did not differ among the 4-time points in non - sirs   patients . 
 conclusions . 
 upregulation of tlr2/4 expression on pbmc may contribute to the development 
 of postoperative sirs during perioperative period of lt .