Comparison of serum biomarkers for the early diagnosis of patients with liver cirrhosis and systemic inflammatory response syndrome

Soluble triggering receptor expressed on myeloid cells-1(sTREM-1),Cytokines,Early Abstract Systemic inflammatory response syndrome (SIRS) can cause serious negative effects among patients with liver cirrhosis (LC). . It is very important to finding methods for early diagnose and intervene early in these patients.This study was to assess the accuracy of early diagnostic value of serum biomarkers in patients with LC and SIRS. A total of 123 LC patients were enrolled, 64 of whom were diagnosed with SIRS and 59 patients without SIRS. Various biomarkers and cytokines were measured in two groups of patients: LC+SIRS and LC−SIRS. Receiver operating characteristic curves (ROCs) were used to assess the ability of tested biomarkers to diagnoseLC with SIRS. C-reactive PCT,


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The WBC count,N% and levels of CRP, PCT, sTREM-1, IL-6, IL-10 and TNF-α are helpful for the early diagnosis of LC+SIRS.Serum sTREM-1 cut-off levels provide better accuracy than customary levels for cirrhosis with SIRS and appears to be a useful early marker to discriminate between SIRS and no-SIRS.It may also be helpful for implications in the prevention and treatment of cirrhosis and SIRS/sepsis.

Background
Patients with liver cirrhosis (LC) have an altered defense against bacteria. This reduced bacterial clearance in LC can contribute to a high risk of systemic inflammatory response syndrome (SIRS) [1]. The latter is a common and serious burden among LC patients because it can cause liver function to deteriorate further, and has serious negative effects on the disease course [2,3]. Some evidence suggests that systemic inflammation in LCpatients maximizes the risk of complications (e.g., portal hypertensive bleeding, hepatic encephalopathy, acute-on-chronic liver failure) and increases the mortality risk due to acute renal insults [3]. About 30% of LC patients die within 1 month of infection and another 30% die within 1 year. We have reported that 90-day mortality in LC patients with SIRS can be up to 38% [4].Therefore, finding methods to diagnose and intervene early in these patients is very important.
Studies have shown that the pathophysiologic background of SIRS and septic complications such as hypercytokinemia, if prolonged, can cause multiple organ dysfunctionsyndrome (MODS) [5]. Cytokines such as interleukin (IL)-6 are thought to be key mediators in the acute response to SIRS and MOF development [6,7].
Cytokines are involved in injury to and cirrhosis of the liver. Clinical research has shown the prominent role of T helper cell type 1 (Th1) cells, Th2 cells, pro-inflammatory or antiinflammatory cytokines in LC pathogenesis [8]. LC patients with SIRS can have acute and extrahepatic manifestations. Cytokines are secreted by immune-system cells and play an 4 important part in infection control, inflammation, regeneration and fibrosis [9]. In recent years, there has been considerable interest in the search for possible immunologic markers for the diagnosis and progression of SIRS/sepsis [10,11]. Knowing the cutoff values for cytokines to predict early SIRS in LC patients would be extremely helpful, but little work has been done in this area. We don't know which is more predictable between levels of serum cytokines and some traditional inflammatory markers such as Creactive protein ( CRP ) and procalcitonin(PCT) in early recognition of cirrhosis with SIRS.
Triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell-surface receptor on blood neutrophils and mature monocytes/macrophages [12]. TREM-1 is a 30-kDa glycoprotein of the immunoglobulin superfamily [12]. In vitroand in vivostudies have shown that TREM-1 expression is upregulated strongly by extracellular bacteria (particularly their cell-wall components) and by fungi [13]. TREM-1 expression can be induced by bacterial products such as lipopolysaccharides and lipoteichoic acid. Recently it is reported that TREM-1 is a potent amplifier of the inflammatory response to invading pathogens because activation of its receptors during infection results in enhanced production of pro-inflammatory cytokines [14]. Hence, TREM-1 has attracted attention as a diagnostic/prognostic biomarker for SIRS/sepsis [14]. A soluble form of TREM-1 (sTREM-1) is released from activated phagocytes and can be found in serum [15]. The plasma level of sTREM-1 appears to be a reliable parameter in differentiating patients with sepsis from those with SIRS [16,17]. However, application of sTREM-1 for the early diagnosis of LC patients with SIRS has not been done.
In the present study, comparison of the white blood cell (WBC) count, neutrophil percentage, as well as levels of CRP,PCT,sTREM-1 and cytokines was done. We wish to find out a effective and sensitive biomarker as a strategy for the early diagnosis of LC patients with SIRS.

Follow-up
Survival data were obtained by telephone contact to patients or their family. All patients in the cohort were studied for 90 days or until death.

Laboratory examination
Routine microbiology examination for SIRS patients involved more than one pair of blood cultures. Analyses of urine, sputum, bronchoalveolar lavage fluid, cerebrospinal fluid, abscesses and closed wounds were undertaken. Blood was drawn immediately after presentation to the Emergency Department of Zhejiang Provincial People's Hospital and analyzed in the laboratory within 24 h for measurement of WBC counts, neutrophil percentage, CRP level, procalcitonin level, and blood chemistry.

Statistical analyses
Descriptive data are the mean ± standard deviation or number (percentage). Comparison of continuous variables was carried out using the Student's t-test. Skewness distribution data are the median with range (25-75 interquartile range) and were analyzed using the Mann-Whitney U-test. Categorical variables were analyzed using Fisher's exact test or Pearson's χ 2 -test. Evaluation of the early diagnostic performance of CRP, procalcitonin, sTREM-1, and cytokines was done using receiver operating characteristic curves (ROCs).
The latter were compared using a non-parametric method. The cutoff value, which was the maximum area under the ROC curve (AUC), and accuracies were calculated with 95% confidence intervals. P < 0.05 was considered significant. SPSS v17.0 (IBM, Armonk, NY, USA) was used for all analyses.

Patient characteristics
Detailed characterization of the 123 patients is shown in Table 1

Comparison of tested biomarkers between the two groups
The WBC count, neutrophil percentage, as well as levels of procalcitonin, CRP, and sTREM-  Table 3.

Discussion
SIRS can occur in patients with liver cirrhosis ofvarious etiology.We found significant differences in levels of ALB, TB,PT,Cr, PLT count, Child-Pugh class C ratio, Child score, infection ratio and 90-day mortality between patients with and without SIRS.LC with SIRS patients were in critical conditionand with a poor in-hospital outcome, especially in patientswith Child-Pugh class C and their lower immunity may be responsible for the high incidence of SIRS.SIRS appears to have important prognostic relevance and increases the risk of encephalopathy, renal failure, infection and death during acuteor chronicliver failure [19,20]. Unfortunately, predicting SIRS early in LC patients is difficult. Serum levels of CRP and procalcitonin have been suggested to be early markers for the diagnosis of SIRS as well as for the diagnosis and prediction of bacterial infection in LC [21,22]. The CRP concentration is closely related to the increasing speed, amplitude, duration, and severity of inflammation.
Previously, we found that CRP >25 mg/L is associated significantly with 90-day mortality in LC+SIRS patients [4].Procalcitonin is a sensitive and specific serologic marker, and its level increases at the early stage of LC with serious bacterial infection, and so has value for early diagnosis [23]. However, our data showed that levels of CRP and procalcitonin in LC+SIRS patients did not increase significantly ( Table 2), even though they were significantly different between the LC+SIRS group and LC-SIRS group. Procalcitonin is derived mostly from the liver. Liver failure may lead to the formation of a network of monocytes to reduce theconcentration of acute-phase proteins (APPs) [24]. The more severe the underlying liver dysfunction, the lower the CRP response to bacteremia [25].
Similarly, the reason why the WBC count and neutrophil percentage were not high may have been related to hypersplenism and decompensated LC. We showed the WBC count was in the normal range even if it was significantly different between LC+SIRS patients and LC-SIRS patients. However,the WBC count was not significantly different in patients who died and those who survived. Some new biomarkers of SIRS would be particularly useful for the early diagnosis in this population.
TREM-1 is expressed on neutrophils and monocytes. It is implicated in the development and amplification of the early inflammatory response to infection and injury. sTREM-1 is the soluble form of this receptor and is released into body fluids when TREM-1 expression is upregulated [26]. As reported by Jedynak and colleagues, the serum level of sTREM-1 measured within the first 24 h of treatment in the intensive care unit is a useful prognostic biomarker for patients with sepsis, severe sepsis or septic shock [27]. sTREM-1 expression is upregulated in the presence of bacteria and fungi, whereas it is expressed only weakly in noninfectious disorders such as vasculitis or psoriasis.
Various studies have suggested that the sTREM-1 concentration in different biologic fluids is significantly higher in patients with bacterial infection than in those with a nonmicrobial inflammatory process [28][29].sTREM-1 is the best biomarker evaluated for the diagnosis and prognosis of sepsis to date [30]. Furthermore, the serum level of sTREM-1 reflects the severity of sepsis more accurately than that of CRP and procalcitonin, and is more sensitive for dynamic evaluation of the sepsis prognosis. [31] The serum concentration of sTREM-1can early predict the 28-day sepsis mortality [32].
We revealed that LC+SIRS patients had a significantly increasedserum level of sTREM-1, the highest AUC (0.940), sensitivity of 0.844, and specificity of 0.942 at a cutoff value of 179.230 ng/mL. Serum levels of sTREM-1 could be used to predict the early diagnosis of LC+SIRS compared with the WBC count, neutrophil percentage or levels of CRP, procalcitonin, IL-6,TNF-or IL-10 (Table 3, Figure 1). Even in the LC+SIRS group, levels of CRP and procalcitonin were more meaningful in the non-survival group than in the survival group. We demonstrated thatthe serum level of sTREM-1 wasa suitable biomarker for identification of LC patients with SIRS. Moreover, the serum level ofsTREM-1 may be an outcome predictor in LC patients with SIRS, and could act as an indicator for starting early therapeutic interventions.
Recenty some data demonstrate that the TREM-1 pathway on Kupffer cells plays an essential role in hepatic inflammation and fibrogenesis in a mouse model of fibrosis.
TREM-1 controls the mobilization of inflammatory cells in response to injury and consequently enhances liver damage [33]. Maybe we will study the relationship between TREM-1 and severity of liver inflammation in the future.
Cytokines are key mediators of pro-and anti-inflammatory processes. LC is associated with impairment of detoxification, synthetic processes, metabolic processes, and alterations (and even increased synthesis) of some pro-inflammatory molecules. The role of the liver as one of the major sites of cytokine production has been acknowledged widely [34]. A severe inflammatory response on the basis of LC can impact directly on circulating levels of cytokines and growth factors and, ultimately, affect immune-system functions [35,36]. Studies have shown that SIRS/sepsis leads to the release of some cytokines, among whichIL-6 and TNF-are important. In addition, levels of IL-4, IL-8, IL-10, IL-12 and interferon-γ are also increased [37,38].

IL-6 release is induced by lipopolysaccharides, viral infections, or products released by necrotic cells. Several scholars have investigated the correlation between levels of IL-6
and TNF-and pathologic states based on inflammatory processes [39,40].
Increasedplasma levels of TNF-have been reported after endotoxin stimulation in healthy volunteers and septic-shock patients with Gram-positive and Gram-negative bacteremia.
The blood concentration of IL-6 and TNF-is positively correlated with the severity of infection and inflammation, and is used as a sensitive indicator to judge disease severity and the prognosis [41].We observed meaningful differences in the concentrations of the pro-inflammatory cytokinesIL-6 and TNF-α, as well as the anti-inflammatory cytokine IL-10, in LC+SIRS patients. However, there were no significant differences in the concentrations of the pro-inflammatory cytokines IL-1β1, IL-2, IL-8, IL-12or IL-17A, or the antiinflammatory cytokine, IL-4 ( Table 2). IL-6 and TNF-α were representative cytokines produced in LC and showed significantly high expression in the early phase of LC+SIRS.
However, their specificity was relatively low for LC+SIRSpatients, and their diagnostic ability inferior to that of sTREM-1.
Our study had two main limitations. First, our study cohort wasrelatively small. Further validation of a larger dataset is required. Second, the serum concentrations of test markers according to the severity of LC with SIRS/sepsis should have been studied. The definition comprises four clinical entities, SIRS, sepsis, severe sepsis and MODS, which may have different early-diagnosis markers and models for prediction of LC [42,43].

Conclusions
The WBC count, neutrophil percentage and levels of CRP, procalcitonin, sTREM-1, IL-6, IL-10 and TNF-α are helpful for the early diagnosis of LC+SIRS, and the serum level of sTREM-1 is a novel biomarker for these patients. Serum sTREM-1 cut-off levels provide better accuracy than customary levels for cirrhosis with SIRS and appears to be a useful early marker to discriminate between LC patients with SIRS and LC patients without SIRS.It may also be helpful for implications in the prevention and treatment of cirrhosis and SIRS/sepsis.    WBC, white blood cell, N%, Neutrophil ratio CRP, C-reactive protein; PCT, procalcitonin; IL, interleukin; TNF--α, tumor necrosis factor-alpha. Figure 1