- Email: [email protected]
High levels of serum mannose-binding lectins are associated with the severity and clinical outcomes of severe traumatic brain injury
Clinica Chimica Acta 451 (2015) 111–116
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
High levels of serum mannose-binding lectins are associated with the severity and clinical outcomes of severe traumatic brain injury Wei Yu a, Hai-Wei Le a, Yi-Gao Lu a, Jun-An Hu a, Jian-Bo Yu b, Ming Wang b, Wei Shen a,⁎ a
Department of Neurosurgery, The People's Hospital of Beilun District, Beilun Branch Hospital of The First Affiliated Hospital of Medical School of Zhejiang University, 1288 Lushan East Road, Beilun District, Ningbo 315800, China Department o f Neurosurgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China
b
a r t i c l e
i n f o
Article history: Received 23 September 2015 Received in revised form 16 October 2015 Accepted 16 October 2015 Available online 23 October 2015 Keywords: Traumatic brain injury Biomarker Complement system Mannose-binding lectin Prognosis Severity
a b s t r a c t Background: Mannose-binding lectin (MBL) is a key component of innate immunity. The expression of cortical MBL is up-regulated after clinical and experimental head trauma. This study aimed to assess the association of serum MBL levels with injury severity and long-term clinical outcomes after severe traumatic brain injury (STBI). Methods: Serum MBL levels were measured in 122 patients and 100 healthy controls. Multivariate analyses were used to analyze the relationship between serum MBL levels and trauma severity reflected by Glasgow Coma Scale scores as well as between serum MBL levels and 6-month mortality and unfavorable outcome (Glasgow Outcome Scale score: 1–3). A receiver operating characteristic (ROC) curve was structured to evaluate the prognostic predictive performance of serum MBL levels. Results: Compared with healthy controls, serum MBL levels of patients were markedly elevated. Using multivariate analyses, serum MBL levels were found to be associated closely with Glasgow Coma Scale (GCS) scores and MBL emerged as an independent predictor for 6-month mortality and unfavorable outcome. Under ROC curve, serum MBL levels and GCS scores possessed similar prognostic predictive values. Conclusion: Increased serum level of MBL was independently associated with head trauma severity and longterm clinical outcomes of STBI. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Severe traumatic brain injury (STBI) is known to be one of the major causes of mortality worldwide and the leading cause of longterm disability as well [1]. The complement system is a major component of the innate immune system [2]. After complement system activation, the sequential production of complement products increases cell injury and death through opsonophagocytosis, cytolysis and inflammatory cell responses [3,4]. Various studies have revealed the wide ranging involvement of complement system in the secondary injury after STB, highlighting the potential for complementtargeted therapeutics to alleviate the devastating consequences of STBI [5,6]. Complement mannose-binding lectin (MBL), a member of the collectin subfamily of C-type lectins, is a key component of innate immunity and activates the complement and promotes opsonophagocytosis [7]. The deficiency of MBL due to several common gene polymorphisms significantly enhances the risk of severe infections, particularly in the neonatal age and in childhood [8]. On the contrary, some studies conclude on the protective role of low levels of MBL toward some
⁎ Corresponding author. E-mail address: [email protected] (W. Shen).
http://dx.doi.org/10.1016/j.cca.2015.10.017 0009-8981/© 2015 Elsevier B.V. All rights reserved.
diseases [9–11]. To our best knowledge, in the central nervous system, except an experimental study showing that MBL gene deficiency increases susceptibility to TBI in mice [12], other studies have demonstrated that low levels of MBL expressions exert the neuroprotective effects in experiment TBI and ischemic stroke [13–16]. Recently, it is verified that the expression of cortical MBL is upregulated after clinical and experimental traumatic brain injury [13]. Some data have shown that high circulating MBL levels are associated with the risk, disease severity and poor clinical outcomes of ischemic stroke [17,18]. However, at present there is a paucity of data available on the change of circulating MBL levels after STBI. 2. Methods 2.1. Participants This prospective, observatory study continuously enrolled the patients with isolated head trauma and postresuscitation Glasgow Coma Scale (GCS) score of 8 or less, who were hospitalized within 6 h since trauma at the People's Hospital of Beilun District during the period from May 2011 to June 2014. Isolated head trauma was defined as head computed tomography scan-confirmed brain injury without other major extracranial injuries, such as pelvis or femur fractures, or severe abdominal or thoracic invasive injuries, as indicated by an
112
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
extracranial abbreviated injury scale score of b3. The current study had excluded the patients with infectious diseases, immunological diseases, use of immunosuppressant, fever within recent 1 month before head trauma, an elevated white blood cell count, positive chest X-ray, b18 y, previous severe head trauma, neurological disease including ischemic or hemorrhagic stroke, use of anti-platelet or anticoagulant medication and presence of other prior systemic diseases including diabetes mellitus, hypertension, uremia, liver cirrhosis, malignancy and chronic heart or lung disease. The control group consisted of healthy volunteers who came to our hospital for healthy examination during the period from June 2013 to June 2014. The study followed the tenets of the Declaration of Helsinki and was approved by the institutional review board of the People's Hospital of Beilun District. The participants or relatives provided written informed consent in advance and potential risks were fully explained. 2.2. Clinical and radiological assessments The recorded information included age, sex, blood pressure, time from trauma to admission, and papillary reactivity. Trauma severity was assessed using postresuscitation GCS scores. Brain lesions were classified according to the Marshall computed tomography criteria [19]. Abnormal cisterns, midline shift at N5 mm and subarachnoid hemorrhage were also recorded after computerized tomography scans. Radiological procedures were completed according to the neuroradiology department protocol. The investigative group comprised a neurosurgeon and a radiologist and they were blinded to clinical information.
(ORs) and 95% confidence intervals (CIs). A receiver operating characteristic (ROC) curve analysis was carried out to test the predictive performance of MBL and area under the curve (AUC) and 95% CI were calculated. Intergroup comparisons of AUCs were performed using the Z test. All statistical analyses were performed with SPSS for Windows, ver 20.0. Statistical significance was defined as P b 0.05. 3. Results 3.1. The subject characteristics During the study period, 158 patients were initially evaluated and 36 patients were excluded because of the reasons listed in Fig. 1. 122 patients were finally included in this study. In addition, 100 healthy controls were recruited in this study. The patients, consisting of 69 men and 53 women, had a mean age of 42.7 ± 17.8 y. The healthy controls, composed of 60 men and 40 women, had a mean age of 40.8 ± 16.1 y. There were not statistically significant differences in gender and age. Among the patients with median initial postresuscitation GCS score of 5 (3), 58 patients (47.5%) had unreactive pupils on admission; 60 patients (49.2%), CT classification 5 or 6; 56 patients (45.9%), abnormal cisterns on initial CT scan; 64 patients (52.5%), midline shift N5 mm on initial CT scan; 68 patients (55.7%), presence of traumatic subarachnoid hemorrhage on initial CT scan; 66 patients (54.1%), and intracranial surgery in 1st 24 h. In terms of mechanism of injury, 69 patients (56.6%) suffered from automobile/motorcycle; 30 patients (24.5%), fall/jump; 23 patients (18.9%), and others. Within 6 months after trauma, 36 patients (29.5%) died from head trauma and 62 patients (50.8%) suffered from unfavorable outcome.
2.3. Clinical outcome evaluation 3.2. The change of serum MBL levels The clinical endpoints for this analysis were mortality and unfavorable outcome within 6 months after admission. The functional outcome was defined by Glasgow Outcome Scale score. Glasgow Outcome Scale was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; and 5 = good recovery [20]. Unfavorable outcome was defined as Glasgow Outcome Scale scores of 1–3. For follow-up, structure telephone interviews were performed by a doctor who was blinded to clinical information.
The admission serum MBL levels were significantly elevated in all patients (1.7 ± 0.7 mg/l), compared with healthy controls (0.7 ± 0.3 mg/l, P b 0.001). Moreover, the serum MBL levels were obviously higher in non-survivors (2.2 ± 0.7 mg/l) than in survivors (1.4 ± 0.5 mg/l, P b 0.001) and in patients with unfavorable outcome (2.0 ± 0.6 mg/l) than in those with favorable outcome (1.3 ± 0.4 mg/l, P b 0.001). 3.3. The correlative analysis
2.4. Assay Venous blood was drawn with minimal stasis through an antecubital vein from patients on admission and from healthy controls at study entry. Clotted blood was centrifuged within 30 min and serum stored at −70 °C until assayed. Microwells coated with europium-labeled anti-MBL antibody were incubated with dilutions of patient serum and europium was quantified with time-resolved immune-fluorometric assay (Baoman Biological Technology Co., Ltd) as described previously [21]. Samples were all processed in duplicate by the same laboratory technician using the same equipment and blinded to all clinical data.
Table 1 showed that the serum MBL levels were highly associated with GCS scores, shock on admission, hyperglycemia on admission, blood oxygen saturation on admission, unreactive pupils, computerized
2.5. Statistical analysis The results were reported as counts (percentage) for the categorical variables, mean ± SD if normally distributed and median (interquartile range) if not normally distributed for the continuous variables. Univariate data were compared by Student's t test or Mann–Whitney Utest for the continuous variables and by χ2 test or Fisher exact test for the categorical variables as appropriate. Correlations were assessed by Spearman correlation coefficient or Pearson correlation coefficient as appropriate. A multivariate linear regression was used further to verify their associations. A multiple binomial logistic regression analysis was performed to identify the association between serum MBL levels and clinical endpoints and the results were reported as odds ratios
Fig. 1. Excluded and included patients with severe traumatic brain injury.
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116 Table 1 The baseline parameters associated with serum mannose-binding lectin levels in the patients with severe traumatic brain injury. Characteristics
r value
P value
Sex (male/female) Age (y) Mechanism of injury GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift N5 mm on initial CT scan Presence of traumatic SAH on initial CT scan Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Mean arterial pressure (mm Hg) Heart rate (beats/min) Body temperature (°C) Respiratory rate (respirations/min) Blood oxygen saturation (percentage) Blood white blood cell count (×109/l) Blood hemoglobin level (g/l) Blood platelet count (×109/l) Blood glucose level (mmol/l) Blood sodium level (mmol/l) Blood potassium level (mmol/l) Prothombin time (s) Thombin time (s) Partial thomboplastin time (s) Plasma C-reactive protein level (mg/l) Plasma fibrinogen level (g/l) Plasma D-dimer level (mg/l)
0.023 0.021 0.100 −0.562 0.199 0.295 0.166 0.175 0.500 0.253 0.309 0.303 0.216 0.116 0.082 0.054 0.149 0.117 0.124 0.034 0.029 0.111 −0.234 0.055 0.033 0.040 0.244 0.055 0.139 0.102 0.085 0.047 0.251 0.038 0.223
NS NS NS b0.001 0.028 0.001 NS NS b0.001 0.005 0.001 0.001 0.017 NS NS NS NS NS NS NS NS NS 0.009 NS NS NS 0.007 NS NS NS NS NS 0.005 NS 0.014
Correlations were assessed by Spearman correlation coefficient or Pearson correlation coefficient as appropriate. GCS indicates Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
tomography classification 5 or 6, abnormal cisterns, midline shift N5 mm, presence of traumatic subarachnoid hemorrhage, blood glucose levels, plasma D-dimer level and plasma C-reactive protein levels. A multivariate linear regression demonstrated that serum MBL levels were still highly associated with GCS scores (t = − 5.143, P b 0.001). This sort of association has been depicted in Fig. 2.
Fig. 2. The relationship between serum mannose-binding lectin (MBL) levels and Glasgow Coma Scale (GCS) scores in patients with severe traumatic brain injury. r value was calculated according to Spearman correlation coefficient.
113
3.4. The prognostic prediction Tables 2 and 3 showed that 6-month mortality and 6-month unfavorable outcome were associated with GCS scores, shock on admission, hyperglycemia on admission, hypoglycemia on admission, hypoxia on admission, unreactive pupils, computerized tomography classification 5 or 6, abnormal cisterns, midline shift N5 mm, presence of traumatic subarachnoid hemorrhage, blood oxygen saturation, blood glucose levels, plasma D-dimer level, plasma C-reactive protein levels and serum MBL levels. When the above variables that the univariate analysis found significant were introduced into the logistic model, GCS scores (OR, 0.297; 95% CI, 0.161–0.547; P b 0.001) and serum MBL levels (OR, 15.807; 95% CI, 3.575–69.907; P b 0.001) emerged as the independent predictors of 6-month mortality of patients, as well as GCS scores (OR, 0.400; 95% CI, 0.281–0.570; P b 0.001) and serum MBL levels (OR, 10.656; 95% CI, 2.875–39.494; P b 0.001) were also identified as the independent predictors of 6-month unfavorable outcome of patients. Under ROC curves, Fig. 3 showed that serum MBL levels predicted 6month mortality and 6-month unfavorable outcome of patients with high AUCs. Based on AUCs, their predictive values were similar to GCS scores' for the prediction of 6-month mortality (AUC, 0.882; 95% CI, 0.811–0.933; P = 0.283) and 6-month unfavorable outcome (AUC, 0.890; 95% CI, 0.820–0.939; P = 0.271).
Table 2 The factors associated with 6-month mortality.
Number Sex (male/female) Age (y) Mechanism of injury Automobile/motorcycle Fall/jump Others GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift N5 mm on initial CT scan Traumatic SAH on initial CT scan Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Mean arterial pressure (mm Hg) Heart rate (beats/min) Body temperature (°C) Respiratory rate (respirations/min) Blood oxygen saturation (percentage) Blood white blood cell count (×109/l) Blood hemoglobin level (g/l) Blood platelet count (×109/l) Blood glucose level (mmol/l) Blood sodium level (mmol/l) Blood potassium level (mmol/l) Prothombin time (s) Thombin time (s) Partial thomboplastin time (s) Plasma C-reactive protein level (mg/l) Plasma fibrinogen level (g/l) Plasma D-dimer level (mg/l) Serum MBL level (mg/l)
Non-survivor
Survivors
36 21/15 43.9 ± 18.4
86 48/38 42.1 ± 17.6
19 13 4 4 (1) 16 (44.4%) 12 (33.3%) 5 (13.9%) 8 (22.2%) 31 (86.1%) 24 (66.7%) 26 (72.2%) 28 (77.8%) 26 (72.2%) 24 (66.7%) 2.5 ± 1.1 3.1 ± 0.9 118.8 ± 35.0 72.3 ± 21.4 87.8 ± 24.4 82.0 ± 20.8 36.3 ± 0.7 18.8 ± 4.6 84.1 ± 9.8 8.0 ± 3.8 128.4 ± 28.4 169.8 ± 45.9 11.9 ± 4.2 142.2 ± 11.0 4.1 ± 1.0 14.8 ± 2.8 19.4 ± 3.1 40.1 ± 5.2 9.4 ± 2.8 3.6 ± 1.3 2.7 ± 1.3 2.2 ± 0.7
50 17 19 7 (3) 8 (9.3%) 14 (16.3%) 3 (3.5%) 6 (7.0%) 27 (31.4%) 36 (41.9%) 30 (34.9%) 36 (41.9%) 42 (48.8%) 42 (48.8%) 2.4 ± 1.5 3.0 ± 1.4 124.0 ± 32.5 69.6 ± 21.4 86.7 ± 22.2 84.7 ± 21.7 36.5 ± 0.8 18.0 ± 3.9 87.9 ± 4.9 7.4 ± 2.3 122.1 ± 22.4 168.9 ± 38.4 10.0 ± 3.2 140.4 ± 7.6 4.0 ± 0.7 14.4 ± 2.5 18.6 ± 3.1 39.3 ± 6.9 8.1 ± 2.1 4.1 ± 1.9 2.3 ± 0.9 1.4 ± 0.5
P value NS NS NS
b0.001 b0.001 0.036 0.048 0.026 b0.001 0.012 b0.001 b0.001 0.018 NS NS NS NS NS NS NS NS NS 0.005 NS NS NS 0.006 NS NS NS NS NS 0.004 NS 0.027 b0.001
Numerical variables were presented as mean ± SD or median (interquartile range) and analyzed by unpaired Student's t test or Mann–Whitney U-test. Categorical variables were expressed as counts (percentage) and analyzed by χ2 test or Fisher exact test. GCS indicates Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage; MBL, mannose-binding lectin.
114
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
Table 3 The factors associated with 6-month unfavorable outcome.
Number Sex (male/female) Age (y) Mechanism of injury Automobile/motorcycle Fall/jump Others GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift N5 mm on initial CT scan Traumatic SAH on initial CT scan Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Mean arterial pressure (mm Hg) Heart rate (beats/min) Body temperature (°C) Respiratory rate (respirations/min) Blood oxygen saturation (percentage) Blood white blood cell count (×109/l) Blood hemoglobin level (g/l) Blood platelet count (×109/l) Blood glucose level (mmol/l) Blood sodium level (mmol/l) Blood potassium level (mmol/l) Prothombin time (s) Thombin time (s) Partial thomboplastin time (s) Plasma C-reactive protein level (mg/l) Plasma fibrinogen level (g/l) Plasma D-dimer level (mg/l) Serum MBL level (mg/l)
Unfavorable outcome
Favorable outcome
62 38/24 45.7 ± 17.8
60 31/29 39.5 ± 17.2
36 17 9 4 (2) 19 (30.7%) 19 (30.7%) 7 (11.3%) 11 (17.7%) 51 (82.3%) 38 (61.3%) 44 (71.0%) 46 (74.2%) 42 (67.7%) 38 (61.3%) 2.2 ± 1.0 3.0 ± 0.9 118.7 ± 37.1 67.3 ± 24.2 84.2 ± 24.8 81.3 ± 20.1 36.4 ± 0.7 17.5 ± 4.4 84.7 ± 8.5 7.3 ± 3.2 123.4 ± 24.9 166.9 ± 38.0 11.3 ± 4.1 141.8 ± 9.1 4.0 ± 0.9 14.5 ± 2.3 19.3 ± 3.1 39.6 ± 6.9 9.1 ± 2.8 4.0 ± 1.6 2.6 ± 1.1 2.0 ± 0.6
33 13 14 7 (2) 5 (8.3%) 7 (11.7%) 1 (1.7%) 3 (5.0%) 7 (11.7%) 22 (36.7%) 12 (20.0%) 18 (30.0%) 26 (43.3%) 28 (46.7%) 2.7 ± 1.6 3.1 ± 1.5 126.4 ± 28.4 73.6 ± 17.6 89.9 ± 20.2 86.6 ± 22.5 36.4 ± 0.8 18.9 ± 3.7 88.9 ± 3.7 7.9 ± 2.3 124.5 ± 24.0 171.5 ± 43.3 9.7 ± 2.8 140.0 ± 8.3 4.0 ± 0.7 14.5 ± 2.4 18.4 ± 3.1 39.5 ± 6.0 7.8 ± 1.6 3.9 ± 1.9 2.2 ± 1.0 1.3 ± 0.4
P value NS NS NS
b0.001 0.002 0.010 NS 0.027 b0.001 0.007 b0.001 b0.001 0.007 NS NS NS NS NS NS NS NS NS 0.001 NS NS NS 0.017 NS NS NS NS NS 0.002 NS 0.018 b0.001
Numerical variables were presented as mean ± SD or median (interquartile range) and analyzed by unpaired Student's t test or Mann–Whitney U-test. Categorical variables were expressed as counts (percentage) and analyzed by χ2 test or Fisher exact test. GCS indicates Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage; MBL, mannose-binding lectin.
4. Discussion Since serum MBL levels were found to be elevated in acute ischemic stroke [17,18], but no research have reported the circulating MBL levels after head trauma. The main findings of the current study were that (1) serum MBL levels were markedly higher in STBI patients than in healthy controls; (2) a multivariate linear regression demonstrated that serum MBL levels were highly associated with GCS scores; (3) MBL emerged as an independent prognostic predictor for long-term clinical outcomes including 6-month mortality and 6month unfavorable outcome according to a binary logistic regression analysis; (4) under a ROC curve, serum MBL levels possessed high predictive values for 6-month mortality and 6-month unfavorable outcome; and (5) MBL's predictive performances (indicated by AUC) were similar to GCS score's for the clinical outcomes. The current results show that MBL is associated with trauma severity and long-term prognosis of STBI, suggesting that MBL may be a good prognostic predictor after STBI. Traumatic injury to brain elicits a complex cascade of pathophysiological events, including hypoxia, ischemia, apoptosis, excitotoxicity and inflammation, all of which damage the integrity of spared neurons and thus accentuate tissue injury beyond the initial site of trauma [22]. The complement system has been identified as a major component of the innate immune system and is also recognized as an important
Fig. 3. Receiver operating characteristic (ROC) curve analysis of serum mannose-binding lectin (MBL) levels for identifying severe traumatic brain injury patients with 6-month mortality (A) and 6-month unfavorable outcome (defined as the Glasgow Outcome Scale scores of 1–3) (B). ROC curves were constructed based on the sensitivity and specificity of the serum MBL levels for identifying clinical outcome. The area under the curve was calculated based on the ROC curves and expressed as 95% confidence interval.
participant in physiology and disease [2]. Complement system results in a self-amplifying cascade of proteolytic reactions through any one of four major identified pathways, including classical pathway, lectin pathway, alternative pathway and extrinsic pathway [3]. Under physiological conditions, circulating complement proteins in peripheral blood do not enter the central nervous system through the blood–brain barrier. However, almost all of the components of complement can be synthesized within the central nervous system [23,24]. Accordingly, complement plays a key role in the promotion neurogenesis and regeneration as well as the progression of pathology in a range of acute and chronic disorders [25–27.] Head trauma induces a rapid and dramatic breakdown of the blood– brain barrier. As a result, the immune-privileged brain parenchyma is exposed to the full force of both innate and adaptive components of the immune system, which includes a massive influx of circulating complement and an increased activation of complement in the injured central nervous system [5]. Although this process is important for the clearance of cellular and myelin debris as well as other molecules that may be inhibitory to wound healing and repair, over-activation of complement can compromise the integrity of neurons and oligodendrocytes in neighboring tissue that was originally spared at the time of impact, thus exacerbating and widening neuropathology [28–30]. The activation of the complement system's lectin pathway is mainly elicited by the MBL, which is a lectin type C of innate immunity that
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
binds to mannan present on the surface of microorganisms and mediates opsonophagocytosis directly or by the activation of the lectin pathway [31,32]. Serum MBL may be the link between the humoral innate immune response and the differential spectrum of severity observed in ill individuals [33–35]. Nevertheless, as an immunomodulator, MBL may be protective or may aggravate the illness depending on its level, physicochemical status, microenvironment, and cytokine profile induced by injury, thus explaining the seeming discrepancy among some studies performed in the animal models with acute brain injury where, except for an experimental study showing that MBL gene deficiency increases susceptibility to TBI in mice, other studies have demonstrated that low levels of MBL expressions exert the neuroprotective effects in experiment TBI and ischemic stroke [12–16]. In humans, polymorphisms within the coding and promoter regions of the MBL2 gene lead to functional MBL deficiency by reduced levels of circulating functional MBL multimers [36]. Regarding the effect of MBL on clinical outcomes of acute brain injury, Cervera et al. [16] could demonstrate a significant association of genetically defined MBL deficiency with favorable outcome after three months, though in a small cohort that included a considerable number (19%) of hemorrhagic stroke patients. Furthermore, MBL deficiency is found to be associated with smaller cerebral infarcts and favorable outcome in ischemic stroke patients receiving conservative treatment [18]. Recently, serum MBL levels are found to independently predict functional outcome and mortality 90 days after ischemic stroke [11]. These results indicate that serum MBL levels may be associated with trauma severity and clinical outcomes of STBI. The current study reported the relationships between serum MBL levels and clinical outcomes of STBI as well as between serum MBL levels and trauma severity reflected by GCS scores using multivariate analysis. Furthermore, the predictive values had been assessed based on ROC curves. The results showed that serum MBL levels were elevated and highly associated with GCS scores as well as MBL could independently predict clinical outcomes of STBI with verified high predictive values indicated by AUCs. Moreover, its discriminative power was in the range of the GCS score. Therefore, the detection of circulating MBL levels can be useful for the prognostic prediction and risk stratification of STBI. 5. Conclusions Serum MBL levels are highly associated with trauma severity, 6month mortality and 6-month unfavorable outcome of STBI patients, thereby suggesting that MBL has the potential to be a useful, complementary biomarker to predict STBI prognosis and assist in risk stratification of STBI patients. Abbreviations GCS MBL STBI
Glasgow Coma Scale mannose-binding lectin severe traumatic brain injury
Acknowledgments The authors thank all staffs in the Department of Neurosurgery, The People's Hospital of Beilun District (Ningbo, China) for their technical support. References [1] L. Odgaard, I. Poulsen, L.P. Kammersgaard, S.P. Johnsen, J.F. Nielsen, Surviving severe traumatic brain injury in Denmark: incidence and predictors of highly specialized rehabilitation, Clin. Epidemiol. 7 (2015) 225–234. [2] M. Nonaka, Evolution of the complement system, Subcell. Biochem. 80 (2014) 31–43.
115
[3] N.S. Merle, S.E. Church, V. Fremeaux-Bacchi, L.T. Roumenina, Complement system part I — molecular mechanisms of activation and regulation, Front. Immunol. 6 (2015) 262. [4] N.S. Merle, R. Noe, L. Halbwachs-Mecarelli, V. Fremeaux-Bacchi, L.T. Roumenina, Complement system part II: role in immunity, Front. Immunol. 6 (2015) 257. [5] P.F. Stahel, M.C. Morganti-Kossmann, T. Kossmann, The role of the complement system in traumatic brain injury, Brain Res. Brain Res. Rev. 27 (1998) 243–256. [6] F. Orsini, D. De Blasio, R. Zangari, E.R. Zanier, M.G. De Simoni, Versatility of the complement system in neuroinflammation, neurodegeneration and brain homeostasis, Front. Cell. Neurosci. 8 (2014) 380. [7] M. Scorza, R. Liguori, A. Elce, F. Salvatore, G. Castaldo, Biological role of mannose binding lectin: from newborns to centenarians, Clin. Chim. Acta (2015) (pii: S0009-8981(15)00139–4). [8] J. Luo, F. Xu, G.J. Lu, H.C. Lin, Z.C. Feng, Low mannose-binding lectin (MBL) levels and MBL genetic polymorphisms associated with the risk of neonatal sepsis: an updated meta-analysis, Early Hum. Dev. 90 (2014) 557–564. [9] Q. Huang, G. Shang, H. Deng, J. Liu, Y. Mei, Y. Xu, High mannose-binding lectin serum levels are associated with diabetic retinopathy in Chinese patients with type 2 diabetes, PLoS One 10 (2015), e0130665. [10] J.E. Fildes, S.M. Shaw, A.H. Walker, M. McAlindon, S.G. Williams, B.G. Keevil, et al., Mannose-binding lectin deficiency offers protection from acute graft rejection after heart transplantation, J. Heart Lung Transplant. 27 (2008) 1353–1356. [11] Z.G. Zhang, C. Wang, J. Wang, Z. Zhang, Y.L. Yang, L. Gao, et al., Prognostic value of mannose-binding lectin: 90-day outcome in patients with acute ischemic stroke, Mol. Neurobiol. 51 (2015) 230–239. [12] P.H. Yager, Z. You, T. Qin, H.H. Kim, K. Takahashi, A.B. Ezekowitz, et al., Mannose binding lectin gene deficiency increases susceptibility to traumatic brain injury in mice, J. Cereb. Blood Flow Metab. 28 (2008) 1030–1039. [13] L. Longhi, F. Orsini, D. De Blasio, S. Fumagalli, F. Ortolano, M. Locatelli, et al., Mannose-binding lectin is expressed after clinical and experimental traumatic brain injury and its deletion is protective, Crit. Care Med. 42 (2014) 1910–1918. [14] X. de la Rosa, A. Cervera, A.K. Kristoffersen, C.P. Valdés, H.M. Varma, C. Justicia, et al., Mannose-binding lectin promotes local microvascular thrombosis after transient brain ischemia in mice, Stroke 45 (2014) 1453–1459. [15] H. Morrison, J. Frye, G. Davis-Gorman, J. Funk, P. McDonagh, G. Stahl, et al., The contribution of mannose binding lectin to reperfusion injury after ischemic stroke, Curr. Neurovasc. Res. 8 (2011) 52–63. [16] A. Cervera, A.M. Planas, C. Justicia, X. Urra, J.C. Jensenius, F. Torres, et al., Genetically-defined deficiency of mannose-binding lectin is associated with protection after experimental stroke in mice and outcome in human stroke, PLoS One 5 (2010), e8433. [17] Z.Y. Wang, Z.R. Sun, L.M. Zhang, The relationship between serum mannosebinding lectin levels and acute ischemic stroke risk, Neurochem. Res. 39 (2014) 248–253. [18] M. Osthoff, M. Katan, F. Fluri, P. Schuetz, R. Bingisser, L. Kappos, et al., Mannosebinding lectin deficiency is associated with smaller infarction size and favorable outcome in ischemic stroke patients, PLoS One 6 (2011), e21338. [19] L.F. Marshall, S.B. Marshall, M.R. Klauber, M.V. Clark, A new classification of head injury based on computerized tomography, J. Neurosurg. 75 (1991) S14–S20. [20] B. Jennett, M. Bond, Assessment of outcome after severe brain damage, Lancet 1 (1975) 480–484. [21] L.Z. Guan, Q. Tong, J. Xu, Elevated serum levels of mannose-binding lectin and diabetic nephropathy in type 2 diabetes, PLoS One 10 (2015), e0119699. [22] F.H. Brennan, A.J. Anderson, S.M. Taylor, T.M. Woodruff, M.J. Ruitenberg, Complement activation in the injured central nervous system: another dual-edged sword? J. Neuroinflammation 9 (2012) 137. [23] T.M. Woodruff, R.R. Ager, A.J. Tenner, P.G. Noakes, S.M. Taylor, The role of the complement system and the activation fragment C5a in the central nervous system, Neruomol. Med. 12 (2010) 179–192. [24] R. Veerhuis, H.M. Nielsen, A.J. Tenner, Complement in the brain, Mol. Immunol. 48 (2011) 1592–1603. [25] H. Harvey, S. Durant, The role of glial cells and the complement system in retinal diseases and Alzheimer's disease: common neural degeneration mechanisms, Exp. Brain Res. 232 (2014) 3363–3377. [26] A.H. Stephan, B.A. Barres, B. Stevens, The complement system: an unexpected role in synaptic pruning during development and disease, Annu. Rev. Neurosci. 35 (2012) 369–389. [27] B. Lettiero, A.J. Andersen, A.C. Hunter, S.M. Moghimi, Complement system and the brain: selected pathologies and avenues toward engineering of neurological nanomedicines, J. Control. Release 161 (2012) 283–289. [28] J.J. Alexander, A.J. Anderson, S.R. Barnum, B. Stevens, A.J. Tenner, The complement cascade: Yin–yang in neuroinflammation-neuro-protection and-degeneration, J. Neurochem. 107 (2008) 1169–1187. [29] M.D. Galvan, S. Luchetti, A.M. Burgos, H.X. Nguyen, M.J. Hooshmand, F.P.T. Hamers, et al., Deficiency in complement C1q improves histological and functional locomotor outcome after spinal cord injury, J. Neurosci. 28 (2008) 13876–13888. [30] T.V. Arumugam, T.M. Woodruff, J.D. Lathia, P.K. Selvaraj, M.P. Mattson, S.M. Taylor, Neuroprotection in stroke by complement inhibition and immunoglobulin therapy, Neuroscience 158 (2009) 1074–1089. [31] W.K. Ip, K. Takahashi, R.A. Ezekowitz, L.M. Stuart, Mannose-binding lectin and innate immunity, Immunol. Rev. 230 (2009) 9–21. [32] R.M. Dommett, N. Klein, M.W. Turner, Mannose-binding lectin in innate immunity: past, present and future, Tissue Antigens 68 (2006) 193–209. [33] M. Osthoff, M.M. Dean, P.N. Baird, A.J. Richardson, M. Daniell, R.H. Guymer, et al., Association study of mannose-binding lectin levels and genetic variants in lectin
116
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
pathway proteins with susceptibility to age-related macular degeneration: a case–control study, PLoS One 10 (2015), e0134107. [34] J. Xue, A.H. Liu, B. Zhao, M. Si, Y.Q. Li, Low levels of mannose-binding lectin at admission increase the risk of adverse neurological outcome in preterm infants: a 1-year follow-up study, J. Matern. Fetal Neonatal Med. 7 (2015) 1–5.
[35] Y. Zhao, W. Lin, Z. Li, J. Lin, S. Wang, C. Zeng, et al., High expression of mannosebinding lectin and the risk of vascular complications of diabetes: evidence from a meta-analysis, Diabetes Technol. Ther. 17 (2015) 490–497. [36] P. Garred, Mannose-binding lectin genetics: from a to Z, Biochem. Soc. Trans. 36 (2008) 1461–1466.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
High levels of serum mannose-binding lectins are associated with the severity and clinical outcomes of severe traumatic brain injury Wei Yu a, Hai-Wei Le a, Yi-Gao Lu a, Jun-An Hu a, Jian-Bo Yu b, Ming Wang b, Wei Shen a,⁎ a
Department of Neurosurgery, The People's Hospital of Beilun District, Beilun Branch Hospital of The First Affiliated Hospital of Medical School of Zhejiang University, 1288 Lushan East Road, Beilun District, Ningbo 315800, China Department o f Neurosurgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China
b
a r t i c l e
i n f o
Article history: Received 23 September 2015 Received in revised form 16 October 2015 Accepted 16 October 2015 Available online 23 October 2015 Keywords: Traumatic brain injury Biomarker Complement system Mannose-binding lectin Prognosis Severity
a b s t r a c t Background: Mannose-binding lectin (MBL) is a key component of innate immunity. The expression of cortical MBL is up-regulated after clinical and experimental head trauma. This study aimed to assess the association of serum MBL levels with injury severity and long-term clinical outcomes after severe traumatic brain injury (STBI). Methods: Serum MBL levels were measured in 122 patients and 100 healthy controls. Multivariate analyses were used to analyze the relationship between serum MBL levels and trauma severity reflected by Glasgow Coma Scale scores as well as between serum MBL levels and 6-month mortality and unfavorable outcome (Glasgow Outcome Scale score: 1–3). A receiver operating characteristic (ROC) curve was structured to evaluate the prognostic predictive performance of serum MBL levels. Results: Compared with healthy controls, serum MBL levels of patients were markedly elevated. Using multivariate analyses, serum MBL levels were found to be associated closely with Glasgow Coma Scale (GCS) scores and MBL emerged as an independent predictor for 6-month mortality and unfavorable outcome. Under ROC curve, serum MBL levels and GCS scores possessed similar prognostic predictive values. Conclusion: Increased serum level of MBL was independently associated with head trauma severity and longterm clinical outcomes of STBI. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Severe traumatic brain injury (STBI) is known to be one of the major causes of mortality worldwide and the leading cause of longterm disability as well [1]. The complement system is a major component of the innate immune system [2]. After complement system activation, the sequential production of complement products increases cell injury and death through opsonophagocytosis, cytolysis and inflammatory cell responses [3,4]. Various studies have revealed the wide ranging involvement of complement system in the secondary injury after STB, highlighting the potential for complementtargeted therapeutics to alleviate the devastating consequences of STBI [5,6]. Complement mannose-binding lectin (MBL), a member of the collectin subfamily of C-type lectins, is a key component of innate immunity and activates the complement and promotes opsonophagocytosis [7]. The deficiency of MBL due to several common gene polymorphisms significantly enhances the risk of severe infections, particularly in the neonatal age and in childhood [8]. On the contrary, some studies conclude on the protective role of low levels of MBL toward some
⁎ Corresponding author. E-mail address: [email protected] (W. Shen).
http://dx.doi.org/10.1016/j.cca.2015.10.017 0009-8981/© 2015 Elsevier B.V. All rights reserved.
diseases [9–11]. To our best knowledge, in the central nervous system, except an experimental study showing that MBL gene deficiency increases susceptibility to TBI in mice [12], other studies have demonstrated that low levels of MBL expressions exert the neuroprotective effects in experiment TBI and ischemic stroke [13–16]. Recently, it is verified that the expression of cortical MBL is upregulated after clinical and experimental traumatic brain injury [13]. Some data have shown that high circulating MBL levels are associated with the risk, disease severity and poor clinical outcomes of ischemic stroke [17,18]. However, at present there is a paucity of data available on the change of circulating MBL levels after STBI. 2. Methods 2.1. Participants This prospective, observatory study continuously enrolled the patients with isolated head trauma and postresuscitation Glasgow Coma Scale (GCS) score of 8 or less, who were hospitalized within 6 h since trauma at the People's Hospital of Beilun District during the period from May 2011 to June 2014. Isolated head trauma was defined as head computed tomography scan-confirmed brain injury without other major extracranial injuries, such as pelvis or femur fractures, or severe abdominal or thoracic invasive injuries, as indicated by an
112
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
extracranial abbreviated injury scale score of b3. The current study had excluded the patients with infectious diseases, immunological diseases, use of immunosuppressant, fever within recent 1 month before head trauma, an elevated white blood cell count, positive chest X-ray, b18 y, previous severe head trauma, neurological disease including ischemic or hemorrhagic stroke, use of anti-platelet or anticoagulant medication and presence of other prior systemic diseases including diabetes mellitus, hypertension, uremia, liver cirrhosis, malignancy and chronic heart or lung disease. The control group consisted of healthy volunteers who came to our hospital for healthy examination during the period from June 2013 to June 2014. The study followed the tenets of the Declaration of Helsinki and was approved by the institutional review board of the People's Hospital of Beilun District. The participants or relatives provided written informed consent in advance and potential risks were fully explained. 2.2. Clinical and radiological assessments The recorded information included age, sex, blood pressure, time from trauma to admission, and papillary reactivity. Trauma severity was assessed using postresuscitation GCS scores. Brain lesions were classified according to the Marshall computed tomography criteria [19]. Abnormal cisterns, midline shift at N5 mm and subarachnoid hemorrhage were also recorded after computerized tomography scans. Radiological procedures were completed according to the neuroradiology department protocol. The investigative group comprised a neurosurgeon and a radiologist and they were blinded to clinical information.
(ORs) and 95% confidence intervals (CIs). A receiver operating characteristic (ROC) curve analysis was carried out to test the predictive performance of MBL and area under the curve (AUC) and 95% CI were calculated. Intergroup comparisons of AUCs were performed using the Z test. All statistical analyses were performed with SPSS for Windows, ver 20.0. Statistical significance was defined as P b 0.05. 3. Results 3.1. The subject characteristics During the study period, 158 patients were initially evaluated and 36 patients were excluded because of the reasons listed in Fig. 1. 122 patients were finally included in this study. In addition, 100 healthy controls were recruited in this study. The patients, consisting of 69 men and 53 women, had a mean age of 42.7 ± 17.8 y. The healthy controls, composed of 60 men and 40 women, had a mean age of 40.8 ± 16.1 y. There were not statistically significant differences in gender and age. Among the patients with median initial postresuscitation GCS score of 5 (3), 58 patients (47.5%) had unreactive pupils on admission; 60 patients (49.2%), CT classification 5 or 6; 56 patients (45.9%), abnormal cisterns on initial CT scan; 64 patients (52.5%), midline shift N5 mm on initial CT scan; 68 patients (55.7%), presence of traumatic subarachnoid hemorrhage on initial CT scan; 66 patients (54.1%), and intracranial surgery in 1st 24 h. In terms of mechanism of injury, 69 patients (56.6%) suffered from automobile/motorcycle; 30 patients (24.5%), fall/jump; 23 patients (18.9%), and others. Within 6 months after trauma, 36 patients (29.5%) died from head trauma and 62 patients (50.8%) suffered from unfavorable outcome.
2.3. Clinical outcome evaluation 3.2. The change of serum MBL levels The clinical endpoints for this analysis were mortality and unfavorable outcome within 6 months after admission. The functional outcome was defined by Glasgow Outcome Scale score. Glasgow Outcome Scale was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; and 5 = good recovery [20]. Unfavorable outcome was defined as Glasgow Outcome Scale scores of 1–3. For follow-up, structure telephone interviews were performed by a doctor who was blinded to clinical information.
The admission serum MBL levels were significantly elevated in all patients (1.7 ± 0.7 mg/l), compared with healthy controls (0.7 ± 0.3 mg/l, P b 0.001). Moreover, the serum MBL levels were obviously higher in non-survivors (2.2 ± 0.7 mg/l) than in survivors (1.4 ± 0.5 mg/l, P b 0.001) and in patients with unfavorable outcome (2.0 ± 0.6 mg/l) than in those with favorable outcome (1.3 ± 0.4 mg/l, P b 0.001). 3.3. The correlative analysis
2.4. Assay Venous blood was drawn with minimal stasis through an antecubital vein from patients on admission and from healthy controls at study entry. Clotted blood was centrifuged within 30 min and serum stored at −70 °C until assayed. Microwells coated with europium-labeled anti-MBL antibody were incubated with dilutions of patient serum and europium was quantified with time-resolved immune-fluorometric assay (Baoman Biological Technology Co., Ltd) as described previously [21]. Samples were all processed in duplicate by the same laboratory technician using the same equipment and blinded to all clinical data.
Table 1 showed that the serum MBL levels were highly associated with GCS scores, shock on admission, hyperglycemia on admission, blood oxygen saturation on admission, unreactive pupils, computerized
2.5. Statistical analysis The results were reported as counts (percentage) for the categorical variables, mean ± SD if normally distributed and median (interquartile range) if not normally distributed for the continuous variables. Univariate data were compared by Student's t test or Mann–Whitney Utest for the continuous variables and by χ2 test or Fisher exact test for the categorical variables as appropriate. Correlations were assessed by Spearman correlation coefficient or Pearson correlation coefficient as appropriate. A multivariate linear regression was used further to verify their associations. A multiple binomial logistic regression analysis was performed to identify the association between serum MBL levels and clinical endpoints and the results were reported as odds ratios
Fig. 1. Excluded and included patients with severe traumatic brain injury.
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116 Table 1 The baseline parameters associated with serum mannose-binding lectin levels in the patients with severe traumatic brain injury. Characteristics
r value
P value
Sex (male/female) Age (y) Mechanism of injury GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift N5 mm on initial CT scan Presence of traumatic SAH on initial CT scan Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Mean arterial pressure (mm Hg) Heart rate (beats/min) Body temperature (°C) Respiratory rate (respirations/min) Blood oxygen saturation (percentage) Blood white blood cell count (×109/l) Blood hemoglobin level (g/l) Blood platelet count (×109/l) Blood glucose level (mmol/l) Blood sodium level (mmol/l) Blood potassium level (mmol/l) Prothombin time (s) Thombin time (s) Partial thomboplastin time (s) Plasma C-reactive protein level (mg/l) Plasma fibrinogen level (g/l) Plasma D-dimer level (mg/l)
0.023 0.021 0.100 −0.562 0.199 0.295 0.166 0.175 0.500 0.253 0.309 0.303 0.216 0.116 0.082 0.054 0.149 0.117 0.124 0.034 0.029 0.111 −0.234 0.055 0.033 0.040 0.244 0.055 0.139 0.102 0.085 0.047 0.251 0.038 0.223
NS NS NS b0.001 0.028 0.001 NS NS b0.001 0.005 0.001 0.001 0.017 NS NS NS NS NS NS NS NS NS 0.009 NS NS NS 0.007 NS NS NS NS NS 0.005 NS 0.014
Correlations were assessed by Spearman correlation coefficient or Pearson correlation coefficient as appropriate. GCS indicates Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage.
tomography classification 5 or 6, abnormal cisterns, midline shift N5 mm, presence of traumatic subarachnoid hemorrhage, blood glucose levels, plasma D-dimer level and plasma C-reactive protein levels. A multivariate linear regression demonstrated that serum MBL levels were still highly associated with GCS scores (t = − 5.143, P b 0.001). This sort of association has been depicted in Fig. 2.
Fig. 2. The relationship between serum mannose-binding lectin (MBL) levels and Glasgow Coma Scale (GCS) scores in patients with severe traumatic brain injury. r value was calculated according to Spearman correlation coefficient.
113
3.4. The prognostic prediction Tables 2 and 3 showed that 6-month mortality and 6-month unfavorable outcome were associated with GCS scores, shock on admission, hyperglycemia on admission, hypoglycemia on admission, hypoxia on admission, unreactive pupils, computerized tomography classification 5 or 6, abnormal cisterns, midline shift N5 mm, presence of traumatic subarachnoid hemorrhage, blood oxygen saturation, blood glucose levels, plasma D-dimer level, plasma C-reactive protein levels and serum MBL levels. When the above variables that the univariate analysis found significant were introduced into the logistic model, GCS scores (OR, 0.297; 95% CI, 0.161–0.547; P b 0.001) and serum MBL levels (OR, 15.807; 95% CI, 3.575–69.907; P b 0.001) emerged as the independent predictors of 6-month mortality of patients, as well as GCS scores (OR, 0.400; 95% CI, 0.281–0.570; P b 0.001) and serum MBL levels (OR, 10.656; 95% CI, 2.875–39.494; P b 0.001) were also identified as the independent predictors of 6-month unfavorable outcome of patients. Under ROC curves, Fig. 3 showed that serum MBL levels predicted 6month mortality and 6-month unfavorable outcome of patients with high AUCs. Based on AUCs, their predictive values were similar to GCS scores' for the prediction of 6-month mortality (AUC, 0.882; 95% CI, 0.811–0.933; P = 0.283) and 6-month unfavorable outcome (AUC, 0.890; 95% CI, 0.820–0.939; P = 0.271).
Table 2 The factors associated with 6-month mortality.
Number Sex (male/female) Age (y) Mechanism of injury Automobile/motorcycle Fall/jump Others GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift N5 mm on initial CT scan Traumatic SAH on initial CT scan Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Mean arterial pressure (mm Hg) Heart rate (beats/min) Body temperature (°C) Respiratory rate (respirations/min) Blood oxygen saturation (percentage) Blood white blood cell count (×109/l) Blood hemoglobin level (g/l) Blood platelet count (×109/l) Blood glucose level (mmol/l) Blood sodium level (mmol/l) Blood potassium level (mmol/l) Prothombin time (s) Thombin time (s) Partial thomboplastin time (s) Plasma C-reactive protein level (mg/l) Plasma fibrinogen level (g/l) Plasma D-dimer level (mg/l) Serum MBL level (mg/l)
Non-survivor
Survivors
36 21/15 43.9 ± 18.4
86 48/38 42.1 ± 17.6
19 13 4 4 (1) 16 (44.4%) 12 (33.3%) 5 (13.9%) 8 (22.2%) 31 (86.1%) 24 (66.7%) 26 (72.2%) 28 (77.8%) 26 (72.2%) 24 (66.7%) 2.5 ± 1.1 3.1 ± 0.9 118.8 ± 35.0 72.3 ± 21.4 87.8 ± 24.4 82.0 ± 20.8 36.3 ± 0.7 18.8 ± 4.6 84.1 ± 9.8 8.0 ± 3.8 128.4 ± 28.4 169.8 ± 45.9 11.9 ± 4.2 142.2 ± 11.0 4.1 ± 1.0 14.8 ± 2.8 19.4 ± 3.1 40.1 ± 5.2 9.4 ± 2.8 3.6 ± 1.3 2.7 ± 1.3 2.2 ± 0.7
50 17 19 7 (3) 8 (9.3%) 14 (16.3%) 3 (3.5%) 6 (7.0%) 27 (31.4%) 36 (41.9%) 30 (34.9%) 36 (41.9%) 42 (48.8%) 42 (48.8%) 2.4 ± 1.5 3.0 ± 1.4 124.0 ± 32.5 69.6 ± 21.4 86.7 ± 22.2 84.7 ± 21.7 36.5 ± 0.8 18.0 ± 3.9 87.9 ± 4.9 7.4 ± 2.3 122.1 ± 22.4 168.9 ± 38.4 10.0 ± 3.2 140.4 ± 7.6 4.0 ± 0.7 14.4 ± 2.5 18.6 ± 3.1 39.3 ± 6.9 8.1 ± 2.1 4.1 ± 1.9 2.3 ± 0.9 1.4 ± 0.5
P value NS NS NS
b0.001 b0.001 0.036 0.048 0.026 b0.001 0.012 b0.001 b0.001 0.018 NS NS NS NS NS NS NS NS NS 0.005 NS NS NS 0.006 NS NS NS NS NS 0.004 NS 0.027 b0.001
Numerical variables were presented as mean ± SD or median (interquartile range) and analyzed by unpaired Student's t test or Mann–Whitney U-test. Categorical variables were expressed as counts (percentage) and analyzed by χ2 test or Fisher exact test. GCS indicates Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage; MBL, mannose-binding lectin.
114
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
Table 3 The factors associated with 6-month unfavorable outcome.
Number Sex (male/female) Age (y) Mechanism of injury Automobile/motorcycle Fall/jump Others GCS score on admission Shock on admission Hyperglycemia on admission Hypoglycemia on admission Hypoxia on admission Pupils unreactive on admission CT classification 5 or 6 Abnormal cisterns on initial CT scan Midline shift N5 mm on initial CT scan Traumatic SAH on initial CT scan Intracranial surgery in 1st 24 h Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mm Hg) Diastolic arterial pressure (mm Hg) Mean arterial pressure (mm Hg) Heart rate (beats/min) Body temperature (°C) Respiratory rate (respirations/min) Blood oxygen saturation (percentage) Blood white blood cell count (×109/l) Blood hemoglobin level (g/l) Blood platelet count (×109/l) Blood glucose level (mmol/l) Blood sodium level (mmol/l) Blood potassium level (mmol/l) Prothombin time (s) Thombin time (s) Partial thomboplastin time (s) Plasma C-reactive protein level (mg/l) Plasma fibrinogen level (g/l) Plasma D-dimer level (mg/l) Serum MBL level (mg/l)
Unfavorable outcome
Favorable outcome
62 38/24 45.7 ± 17.8
60 31/29 39.5 ± 17.2
36 17 9 4 (2) 19 (30.7%) 19 (30.7%) 7 (11.3%) 11 (17.7%) 51 (82.3%) 38 (61.3%) 44 (71.0%) 46 (74.2%) 42 (67.7%) 38 (61.3%) 2.2 ± 1.0 3.0 ± 0.9 118.7 ± 37.1 67.3 ± 24.2 84.2 ± 24.8 81.3 ± 20.1 36.4 ± 0.7 17.5 ± 4.4 84.7 ± 8.5 7.3 ± 3.2 123.4 ± 24.9 166.9 ± 38.0 11.3 ± 4.1 141.8 ± 9.1 4.0 ± 0.9 14.5 ± 2.3 19.3 ± 3.1 39.6 ± 6.9 9.1 ± 2.8 4.0 ± 1.6 2.6 ± 1.1 2.0 ± 0.6
33 13 14 7 (2) 5 (8.3%) 7 (11.7%) 1 (1.7%) 3 (5.0%) 7 (11.7%) 22 (36.7%) 12 (20.0%) 18 (30.0%) 26 (43.3%) 28 (46.7%) 2.7 ± 1.6 3.1 ± 1.5 126.4 ± 28.4 73.6 ± 17.6 89.9 ± 20.2 86.6 ± 22.5 36.4 ± 0.8 18.9 ± 3.7 88.9 ± 3.7 7.9 ± 2.3 124.5 ± 24.0 171.5 ± 43.3 9.7 ± 2.8 140.0 ± 8.3 4.0 ± 0.7 14.5 ± 2.4 18.4 ± 3.1 39.5 ± 6.0 7.8 ± 1.6 3.9 ± 1.9 2.2 ± 1.0 1.3 ± 0.4
P value NS NS NS
b0.001 0.002 0.010 NS 0.027 b0.001 0.007 b0.001 b0.001 0.007 NS NS NS NS NS NS NS NS NS 0.001 NS NS NS 0.017 NS NS NS NS NS 0.002 NS 0.018 b0.001
Numerical variables were presented as mean ± SD or median (interquartile range) and analyzed by unpaired Student's t test or Mann–Whitney U-test. Categorical variables were expressed as counts (percentage) and analyzed by χ2 test or Fisher exact test. GCS indicates Glasgow Coma Scale; CT, computerized tomography; SAH, subarachnoid hemorrhage; MBL, mannose-binding lectin.
4. Discussion Since serum MBL levels were found to be elevated in acute ischemic stroke [17,18], but no research have reported the circulating MBL levels after head trauma. The main findings of the current study were that (1) serum MBL levels were markedly higher in STBI patients than in healthy controls; (2) a multivariate linear regression demonstrated that serum MBL levels were highly associated with GCS scores; (3) MBL emerged as an independent prognostic predictor for long-term clinical outcomes including 6-month mortality and 6month unfavorable outcome according to a binary logistic regression analysis; (4) under a ROC curve, serum MBL levels possessed high predictive values for 6-month mortality and 6-month unfavorable outcome; and (5) MBL's predictive performances (indicated by AUC) were similar to GCS score's for the clinical outcomes. The current results show that MBL is associated with trauma severity and long-term prognosis of STBI, suggesting that MBL may be a good prognostic predictor after STBI. Traumatic injury to brain elicits a complex cascade of pathophysiological events, including hypoxia, ischemia, apoptosis, excitotoxicity and inflammation, all of which damage the integrity of spared neurons and thus accentuate tissue injury beyond the initial site of trauma [22]. The complement system has been identified as a major component of the innate immune system and is also recognized as an important
Fig. 3. Receiver operating characteristic (ROC) curve analysis of serum mannose-binding lectin (MBL) levels for identifying severe traumatic brain injury patients with 6-month mortality (A) and 6-month unfavorable outcome (defined as the Glasgow Outcome Scale scores of 1–3) (B). ROC curves were constructed based on the sensitivity and specificity of the serum MBL levels for identifying clinical outcome. The area under the curve was calculated based on the ROC curves and expressed as 95% confidence interval.
participant in physiology and disease [2]. Complement system results in a self-amplifying cascade of proteolytic reactions through any one of four major identified pathways, including classical pathway, lectin pathway, alternative pathway and extrinsic pathway [3]. Under physiological conditions, circulating complement proteins in peripheral blood do not enter the central nervous system through the blood–brain barrier. However, almost all of the components of complement can be synthesized within the central nervous system [23,24]. Accordingly, complement plays a key role in the promotion neurogenesis and regeneration as well as the progression of pathology in a range of acute and chronic disorders [25–27.] Head trauma induces a rapid and dramatic breakdown of the blood– brain barrier. As a result, the immune-privileged brain parenchyma is exposed to the full force of both innate and adaptive components of the immune system, which includes a massive influx of circulating complement and an increased activation of complement in the injured central nervous system [5]. Although this process is important for the clearance of cellular and myelin debris as well as other molecules that may be inhibitory to wound healing and repair, over-activation of complement can compromise the integrity of neurons and oligodendrocytes in neighboring tissue that was originally spared at the time of impact, thus exacerbating and widening neuropathology [28–30]. The activation of the complement system's lectin pathway is mainly elicited by the MBL, which is a lectin type C of innate immunity that
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
binds to mannan present on the surface of microorganisms and mediates opsonophagocytosis directly or by the activation of the lectin pathway [31,32]. Serum MBL may be the link between the humoral innate immune response and the differential spectrum of severity observed in ill individuals [33–35]. Nevertheless, as an immunomodulator, MBL may be protective or may aggravate the illness depending on its level, physicochemical status, microenvironment, and cytokine profile induced by injury, thus explaining the seeming discrepancy among some studies performed in the animal models with acute brain injury where, except for an experimental study showing that MBL gene deficiency increases susceptibility to TBI in mice, other studies have demonstrated that low levels of MBL expressions exert the neuroprotective effects in experiment TBI and ischemic stroke [12–16]. In humans, polymorphisms within the coding and promoter regions of the MBL2 gene lead to functional MBL deficiency by reduced levels of circulating functional MBL multimers [36]. Regarding the effect of MBL on clinical outcomes of acute brain injury, Cervera et al. [16] could demonstrate a significant association of genetically defined MBL deficiency with favorable outcome after three months, though in a small cohort that included a considerable number (19%) of hemorrhagic stroke patients. Furthermore, MBL deficiency is found to be associated with smaller cerebral infarcts and favorable outcome in ischemic stroke patients receiving conservative treatment [18]. Recently, serum MBL levels are found to independently predict functional outcome and mortality 90 days after ischemic stroke [11]. These results indicate that serum MBL levels may be associated with trauma severity and clinical outcomes of STBI. The current study reported the relationships between serum MBL levels and clinical outcomes of STBI as well as between serum MBL levels and trauma severity reflected by GCS scores using multivariate analysis. Furthermore, the predictive values had been assessed based on ROC curves. The results showed that serum MBL levels were elevated and highly associated with GCS scores as well as MBL could independently predict clinical outcomes of STBI with verified high predictive values indicated by AUCs. Moreover, its discriminative power was in the range of the GCS score. Therefore, the detection of circulating MBL levels can be useful for the prognostic prediction and risk stratification of STBI. 5. Conclusions Serum MBL levels are highly associated with trauma severity, 6month mortality and 6-month unfavorable outcome of STBI patients, thereby suggesting that MBL has the potential to be a useful, complementary biomarker to predict STBI prognosis and assist in risk stratification of STBI patients. Abbreviations GCS MBL STBI
Glasgow Coma Scale mannose-binding lectin severe traumatic brain injury
Acknowledgments The authors thank all staffs in the Department of Neurosurgery, The People's Hospital of Beilun District (Ningbo, China) for their technical support. References [1] L. Odgaard, I. Poulsen, L.P. Kammersgaard, S.P. Johnsen, J.F. Nielsen, Surviving severe traumatic brain injury in Denmark: incidence and predictors of highly specialized rehabilitation, Clin. Epidemiol. 7 (2015) 225–234. [2] M. Nonaka, Evolution of the complement system, Subcell. Biochem. 80 (2014) 31–43.
115
[3] N.S. Merle, S.E. Church, V. Fremeaux-Bacchi, L.T. Roumenina, Complement system part I — molecular mechanisms of activation and regulation, Front. Immunol. 6 (2015) 262. [4] N.S. Merle, R. Noe, L. Halbwachs-Mecarelli, V. Fremeaux-Bacchi, L.T. Roumenina, Complement system part II: role in immunity, Front. Immunol. 6 (2015) 257. [5] P.F. Stahel, M.C. Morganti-Kossmann, T. Kossmann, The role of the complement system in traumatic brain injury, Brain Res. Brain Res. Rev. 27 (1998) 243–256. [6] F. Orsini, D. De Blasio, R. Zangari, E.R. Zanier, M.G. De Simoni, Versatility of the complement system in neuroinflammation, neurodegeneration and brain homeostasis, Front. Cell. Neurosci. 8 (2014) 380. [7] M. Scorza, R. Liguori, A. Elce, F. Salvatore, G. Castaldo, Biological role of mannose binding lectin: from newborns to centenarians, Clin. Chim. Acta (2015) (pii: S0009-8981(15)00139–4). [8] J. Luo, F. Xu, G.J. Lu, H.C. Lin, Z.C. Feng, Low mannose-binding lectin (MBL) levels and MBL genetic polymorphisms associated with the risk of neonatal sepsis: an updated meta-analysis, Early Hum. Dev. 90 (2014) 557–564. [9] Q. Huang, G. Shang, H. Deng, J. Liu, Y. Mei, Y. Xu, High mannose-binding lectin serum levels are associated with diabetic retinopathy in Chinese patients with type 2 diabetes, PLoS One 10 (2015), e0130665. [10] J.E. Fildes, S.M. Shaw, A.H. Walker, M. McAlindon, S.G. Williams, B.G. Keevil, et al., Mannose-binding lectin deficiency offers protection from acute graft rejection after heart transplantation, J. Heart Lung Transplant. 27 (2008) 1353–1356. [11] Z.G. Zhang, C. Wang, J. Wang, Z. Zhang, Y.L. Yang, L. Gao, et al., Prognostic value of mannose-binding lectin: 90-day outcome in patients with acute ischemic stroke, Mol. Neurobiol. 51 (2015) 230–239. [12] P.H. Yager, Z. You, T. Qin, H.H. Kim, K. Takahashi, A.B. Ezekowitz, et al., Mannose binding lectin gene deficiency increases susceptibility to traumatic brain injury in mice, J. Cereb. Blood Flow Metab. 28 (2008) 1030–1039. [13] L. Longhi, F. Orsini, D. De Blasio, S. Fumagalli, F. Ortolano, M. Locatelli, et al., Mannose-binding lectin is expressed after clinical and experimental traumatic brain injury and its deletion is protective, Crit. Care Med. 42 (2014) 1910–1918. [14] X. de la Rosa, A. Cervera, A.K. Kristoffersen, C.P. Valdés, H.M. Varma, C. Justicia, et al., Mannose-binding lectin promotes local microvascular thrombosis after transient brain ischemia in mice, Stroke 45 (2014) 1453–1459. [15] H. Morrison, J. Frye, G. Davis-Gorman, J. Funk, P. McDonagh, G. Stahl, et al., The contribution of mannose binding lectin to reperfusion injury after ischemic stroke, Curr. Neurovasc. Res. 8 (2011) 52–63. [16] A. Cervera, A.M. Planas, C. Justicia, X. Urra, J.C. Jensenius, F. Torres, et al., Genetically-defined deficiency of mannose-binding lectin is associated with protection after experimental stroke in mice and outcome in human stroke, PLoS One 5 (2010), e8433. [17] Z.Y. Wang, Z.R. Sun, L.M. Zhang, The relationship between serum mannosebinding lectin levels and acute ischemic stroke risk, Neurochem. Res. 39 (2014) 248–253. [18] M. Osthoff, M. Katan, F. Fluri, P. Schuetz, R. Bingisser, L. Kappos, et al., Mannosebinding lectin deficiency is associated with smaller infarction size and favorable outcome in ischemic stroke patients, PLoS One 6 (2011), e21338. [19] L.F. Marshall, S.B. Marshall, M.R. Klauber, M.V. Clark, A new classification of head injury based on computerized tomography, J. Neurosurg. 75 (1991) S14–S20. [20] B. Jennett, M. Bond, Assessment of outcome after severe brain damage, Lancet 1 (1975) 480–484. [21] L.Z. Guan, Q. Tong, J. Xu, Elevated serum levels of mannose-binding lectin and diabetic nephropathy in type 2 diabetes, PLoS One 10 (2015), e0119699. [22] F.H. Brennan, A.J. Anderson, S.M. Taylor, T.M. Woodruff, M.J. Ruitenberg, Complement activation in the injured central nervous system: another dual-edged sword? J. Neuroinflammation 9 (2012) 137. [23] T.M. Woodruff, R.R. Ager, A.J. Tenner, P.G. Noakes, S.M. Taylor, The role of the complement system and the activation fragment C5a in the central nervous system, Neruomol. Med. 12 (2010) 179–192. [24] R. Veerhuis, H.M. Nielsen, A.J. Tenner, Complement in the brain, Mol. Immunol. 48 (2011) 1592–1603. [25] H. Harvey, S. Durant, The role of glial cells and the complement system in retinal diseases and Alzheimer's disease: common neural degeneration mechanisms, Exp. Brain Res. 232 (2014) 3363–3377. [26] A.H. Stephan, B.A. Barres, B. Stevens, The complement system: an unexpected role in synaptic pruning during development and disease, Annu. Rev. Neurosci. 35 (2012) 369–389. [27] B. Lettiero, A.J. Andersen, A.C. Hunter, S.M. Moghimi, Complement system and the brain: selected pathologies and avenues toward engineering of neurological nanomedicines, J. Control. Release 161 (2012) 283–289. [28] J.J. Alexander, A.J. Anderson, S.R. Barnum, B. Stevens, A.J. Tenner, The complement cascade: Yin–yang in neuroinflammation-neuro-protection and-degeneration, J. Neurochem. 107 (2008) 1169–1187. [29] M.D. Galvan, S. Luchetti, A.M. Burgos, H.X. Nguyen, M.J. Hooshmand, F.P.T. Hamers, et al., Deficiency in complement C1q improves histological and functional locomotor outcome after spinal cord injury, J. Neurosci. 28 (2008) 13876–13888. [30] T.V. Arumugam, T.M. Woodruff, J.D. Lathia, P.K. Selvaraj, M.P. Mattson, S.M. Taylor, Neuroprotection in stroke by complement inhibition and immunoglobulin therapy, Neuroscience 158 (2009) 1074–1089. [31] W.K. Ip, K. Takahashi, R.A. Ezekowitz, L.M. Stuart, Mannose-binding lectin and innate immunity, Immunol. Rev. 230 (2009) 9–21. [32] R.M. Dommett, N. Klein, M.W. Turner, Mannose-binding lectin in innate immunity: past, present and future, Tissue Antigens 68 (2006) 193–209. [33] M. Osthoff, M.M. Dean, P.N. Baird, A.J. Richardson, M. Daniell, R.H. Guymer, et al., Association study of mannose-binding lectin levels and genetic variants in lectin
116
W. Yu et al. / Clinica Chimica Acta 451 (2015) 111–116
pathway proteins with susceptibility to age-related macular degeneration: a case–control study, PLoS One 10 (2015), e0134107. [34] J. Xue, A.H. Liu, B. Zhao, M. Si, Y.Q. Li, Low levels of mannose-binding lectin at admission increase the risk of adverse neurological outcome in preterm infants: a 1-year follow-up study, J. Matern. Fetal Neonatal Med. 7 (2015) 1–5.
[35] Y. Zhao, W. Lin, Z. Li, J. Lin, S. Wang, C. Zeng, et al., High expression of mannosebinding lectin and the risk of vascular complications of diabetes: evidence from a meta-analysis, Diabetes Technol. Ther. 17 (2015) 490–497. [36] P. Garred, Mannose-binding lectin genetics: from a to Z, Biochem. Soc. Trans. 36 (2008) 1461–1466.