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Increased serum concentrations of transforming growth factor-β1 (TGF-β1) in patients with Guillain-Barré syndrome
Clinica Chimica Acta 461 (2016) 8–13
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Increased serum concentrations of transforming growth factor-β1 (TGF-β1) in patients with Guillain-Barré syndrome Kuo-Hsuan Chang, Rong-Kuo Lyu, Yen-Shi Ro, Yi-Chun Chen, Long-Sun Ro, Hong-Shiu Chang, Ching-Chang Huang, Ming-Feng Liao, Yih-Ru Wu, Hong-Chou Kuo, Chun-Che Chu, Chiung-Mei Chen ⁎ Department of Neurology, Chang Gung Memorial Hospital-Linkou Medical Center, Chang Gung University College of Medicine, Taoyuan, Taiwan
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Article history: Received 12 April 2016 Received in revised form 19 July 2016 Accepted 19 July 2016 Available online 20 July 2016 Keywords: Guillain-Barré syndrome TGF-β1 Biomarker
a b s t r a c t Background: Guillain-Barré syndrome (GBS) is an acquired demyelinating peripheral neuropathy. It has shown that macrophage activation contribute to the pathogenesis of GBS. Therefore macrophage-mediated factors could be the potential markers for disease diagnosis and status of GBS. Methods: We measured serum concentrations of 4 macrophage-mediated factors, including interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), vascular cell adhesion protein 1 (VCAM-1) and vascular endothelial growth factor (VEGF), in 23 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), 28 GBS, 11 Miller-Fisher syndrome (MFS), 40 multiple sclerosis (MS), and 12 Alzheimer's disease (AD) patients, as well as 15 healthy controls. Results: Serum TGF-β1 concentration of GBS patients (35.94 ± 2.55 ng/ml) was significantly higher compared with CIDP (25.46 ± 1.40 ng/ml, P b 0.001), MFS (25.32 ± 2.31 ng/ml, P = 0.010), MS (21.35 ± 0.90 ng/ml, P b 0.001) and AD patients (22.92 ± 1.82 ng/ml, P b 0.001), as well as healthy controls (23.12 ± 1.67 ng/ml, P b 0.001). A positive correlation between serum TGF-β1 concentrations and Hughes' functional grading scales was observed in GBS patients. Serum concentrations of IL-6, VCAM-1 and VEGF were similar between the studied groups. Conclusion: The high serum concentrations of TGF-β1 and the correlation between serum TGF-β1 concentration and disease severity highlight the potential of TGF-β1 as a biomarker of GBS. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Guillain-Barré syndrome (GBS) is an acquired acute inflammatory peripheral neuropathies [1]. According to different pathophysiological and clinical features, GBS can be classified into several subtypes such as acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), and Miller-Fisher syndrome (MFS) [1]. GBS shares many symptoms and signs with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) particularly in the acute phase of disease. The time to nadir and the subsequent course of the disease
Abbreviations: AD, Alzheimer's disease; AIDP, acute inflammatory demyelinating polyneuropathy; AMAN, acute motor axonal neuropathy; AMSAN, acute motor and sensory axonal neuropathy; CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; CSF, cerebrospinal fluid; GBS, Guillain-Barré syndrome; IL-6, interleukin-6; INCAT disability scale, inflammatory neuropathy cause and treatment disability scale; MFS, Miller-Fisher syndrome; MS, multiple sclerosis; TGF-β1, transforming growth factor-β1; VCAM-1, vascular cell adhesion protein 1; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Department of Neurology, Chang Gung Memorial HospitalLinkou Medical Center, No.5, Fusing St., Gueishan Township, Taoyuan County 333, Taiwan. E-mail address: [email protected] (C.-M. Chen).
http://dx.doi.org/10.1016/j.cca.2016.07.013 0009-8981/© 2016 Elsevier B.V. All rights reserved.
are important clinical differences between these 2 diseases. GBS is a monophasic disease in which the time to reach nadir by definition is within 4 weeks [1], whereas the initial progressive phase lasts N 2 months will be considered as CIDP [2]. The diagnosis of GBS is rather based on a combination of clinical, electrophysiological features as well as analysis of the cerebrospinal fluid (CSF) at present [1]. However, the electrophysiological and CSF examinations fail to show the abnormalities at the early stage of GBS [1]. A biomarker that can be evaluated as an indicator of a pathological process or pharmacological response to a therapeutic intervention could assist in the clinical diagnosis, monitoring disease progression, and testing the efficacy of immunotherapy in GBS. The pathogenesis of GBS is thought to be immune-mediated [3]. This immune response, possibly triggered by antecedent infection [1], may generate antibodies that cross-react with gangliosides at myelin, resulting in slowness or blockade of nerve conduction as well as damage of axons by inflammatory infiltrates [4]. Given that inflammation in GBS could be driven by peripheral lymphocytes, discovery of GBS-specific inflammatory biomarkers in peripheral blood has been repeatedly attempted. So far, a list of GBS-associated biomarkers including haptoglobin [5,6], prealbumin [7], interferon-γ [8], tumor necrosis factor-α [8], interleukin (IL)-17 [9], IL-18 [10], IL-22 [9], IL-37 [8], soluble C5b-9
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complex complement [11], matrix metalloproteinase-9 [12] and neurofilaments [13] has been disclosed in blood or CSF samples. All of these results support the critical role of peripheral inflammation in the pathogenesis GBS. Pathologically, the inflammation of GBS may not be limited to peripheral nerves. Inflammatory infiltrates are also found in spinal cord of GBS patients [14]. The major part of inflammatory cells in GBS, activated macrophage [15], can secrete cytokines, such as IL-6 [16], transforming growth factor-β1 (TGF-β1) [16], vascular cell adhesion protein 1 (VCAM-1) [17], and vascular endothelial growth factor (VEGF) [16], which are usually associated with pro-inflammatory or anti-inflammatory responses. 2. Subjects and methods 2.1. Ethics statement This study was performed under a protocol approved by the institutional review boards of Chang Gung Memorial Hospital (ethical license No: 103-4555C) and all examinations were performed after obtaining written informed consents. 2.2. Sample collection Serum samples were collected from 23 CIDP (10 females, 13 males), 28 GBS (15 females, 13 males, 11 MFS (5 females, 6 males), 40 MS (28 females, 12 males), and 12 AD (7 females, 5 males) patients, as well as 15 healthy controls (7 females, 8 males). The diagnosis of GBS [18], CIDP [19], MFS [20], MS [21] and AD [22] were made according to respective diagnostic criteria. The neurological disability of the patients at the time of nadir was assessed using the Hughes' functional grading scale (for patients with GBS and MFS) [23] or Inflammatory Neuropathy Cause and Treatment (INCAT) disability scale (for patients with CIDP) [24] on the basis of the results of neurological examination and ambulatory ability assessment. All subjects were confirmed to have no systemic infection, chronic renal failure, cardiac or liver dysfunction, malignancies, or autoimmune diseases other than GBS, CIDP, MFS, MS and AD. In the patients with GBS, MFS and MS, serum and CSF samples were collected within 2 weeks after the onset or during acute relapses of diseases. All samples were obtained before treatment with plasmapheresis, or intravenous immunoglobulins. Sample analyses were blindly performed with respect to patients' diagnosis and the results of other tests from the same patient. Serum samples were kept sitting at 4 °C for 4 h and then centrifuged, aliquoted, frozen at − 80 °C, and stored until analysis. The albumin and immunoglobulin (IgG) concentrations in serum and CSF were measured by the Department of Clinical Pathology, Chang Gung Memorial Hospital. 2.3. Anti-ganglioside and anti-neuronal antigen antibodies assay Anti-ganglioside (anti-GD1a, anti-GD1b, anti-GM1, anti-GM2, antiGM3, anti-GQ1b and anti-GT1b) and anti-neuronal antigen (antiAMPHIPHYSIN, anti-CV2, anti-Hu, anti-Ma2, anti-RECOVERIN, anti-Ri, anti-SOX1, anti-TITIN and anti-Yo) IgG autoantibodies were measured by using immunoblot strip kit (Euroimmun AG) according to the manufacturer's instruction. 2.4. Enzyme-linked immunosorbent assays for quantification of targeted inflammatory markers Serum concentrations of IL-6, TGF-β1, VEGF and VCAM-1 were assessed using enzyme-linked immunosorbent assay (ELISA) (R & D Systems). Each assay was performed in duplicate according to the manufacturer's instruction.
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2.5. Statistical analysis The Statistical Program for Social Sciences (SPSS) was used to analyze all the statistics. Non-categorical variables were compared using the Student's t-test, 1-way analysis of variance with Bonferroni post hoc test. Generalized linear model with adjustment for age and gender was applied to evaluate the correlation between serum TGF-β1 concentrations and the Hughes' functional grading scale or INCAT disability scale at the time of sample collection. Each set of data was expressed as mean ± standard error. All P values were 2-tailed, and a P b 0.05 was considered significant. Receiver operating characteristic curve analysis was used to measure the ability of TGF-β1 concentration to predict the diagnosis of GBS. 3. Results The demographic data of all groups were displayed in Table 1. Not surprisingly, the age of the patients with AD (75.7 ± 1.63 y) was significantly higher than the other groups (P b 0.001). IgG index in the patient with MS (1.02 ± 0.05) was significant higher compared with those with CIDP (0.60 ± 0.07, P = 0.002), GBS (0.69 ± 0.05, P = 0.001) and MFS (0.61 ± 0.15, P = 0.026). A panel of serum anti-ganglionside IgG auto-antibodies including anti-GM1, anti-GM2, anti-GM3, anti-GD1a, anti-GD1b, anti-GT1b and anti-GQ1b were measured for these patients. The results showed a number of CIDP patients had anti-GT1b (34.78%), anti-GM3 (30.43%) and anti-GD1b (26.09%). These auto-antibodies were present in fewer patients with GBS compared with those with CIDP, although the differences did not reach the statistical significance. Anti-GQ1b, thought as a diagnostic marker for MFS, was significantly increased (45.45%) in patients with MFS when compared with the other groups (P = 0.008–0.048). Paraneoplastic anti-neuronal antibodies including anti-AMPHIPHYSIN, anti-CV2, anti-Hu, anti-Ma2, antiRECOVERIN, anti-Ri, anti-SOX1, anti-TITIN and anti-Yo were negative in all patients. Activation of macrophage plays an important role in the peripheral neuroinflammatory diseases. By examining four macrophage-mediated inflammatory factors, IL-6, TGF-β1, VCAM-1 and VEGF, in the patients with CIDP, GBS, MFS, MS, and AD, as well as healthy controls, we found serum concentrations of TGF-β1 were significantly increased in GBS patients (35.94 ± 2.55 ng/ml, Fig. 1A) compared to those with CIDP (25.46 ± 1.40 ng/ml, P b 0.001), MFS (25.32 ± 2.31 ng/ml, P = 0.010), MS (21.35 ± 0.90 ng/ml, P b 0.001) and AD (22.92 ± 1.82 ng/ ml, P b 0.001), as well as healthy controls (23.12 ± 1.67 ng/ml, P b 0.001). The area under ROC curve (AUC) for TGF-β1 was 0.78. None of the other three markers showed significant differences between the six groups (Fig. 1B–D). We analyzed the correlation between the serum concentrations of TGF-β1 and disease severity that was evaluated by scores of Hughes' functional grading scale (GBS and MFS) or INCAT disability scale (CIDP) at the time point of sample collection. The results showed a significant correlation between serum concentrations of TGF-β1 and Hughes' functional grading scale in the patients with GBS (β coefficient: 6.05 ± 1.26 ng/ml, P b 0.001, Fig. 2A and Table 2). On the other hand, such clinical correlation is absent in the patients with MFS and CIDP (Fig. 2B and C, Table 2). The CSF concentration of TGF-β1 was undetectable by the assay we used. 4. Discussion Given that a peripheral nervous tissue sample from GBS patients is practically difficult to access, biomarkers in blood should be more feasible as an indicator for the disease severity as well as testing potential therapeutic strategies. In the present study, we demonstrated that serum TGF-β1 concentration was higher in the GBS patients when compared with MFS, CIDP and AD patients, as well as controls. Furthermore, a significant correlation between serum TGF-β1 concentrations and Hughes' functional grading scale scores was demonstrated in GBS
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Table 1 Clinical characteristics of the patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain-Barré syndrome (GBS), Miller-Fisher syndrome (MFS), multiple sclerosis (MS), Alzheimer's disease (AD) and healthy controls (HC). Parameter
CIDP (n = 23)
GBS (n = 28)
MFS (n = 11)
MS (n = 40)
AD (n = 12)
HC (n = 15)
Gender (male/female) Age (y) (range) Hughes' functional grading scale (range) INCAT disability scale (range) IgG index Anti-GD1a IgG (%) Anti-GD1b IgG (%) Anti-GM1 IgG (%) Anti-GM2 IgG (%) Anti-GM3 IgG (%) Anti-GQ1b IgG (%) Anti-GT1b IgG (%)
13/10 50.0 ± 2.9 (22–80)
13/15 47.0 ± 3.4 (27–83) 2.21 ± 1.52 (1–5)
6/5 43.1 ± 4.8 (18–65) 1.82 ± 0.98 (1–3)
12/28 37.3 ± 1.8 (19–65)
5/7 75.7 ± 1.6⁎ (63–84)
8/7 47.0 ± 156 (27–84)
0.69 ± 0.05 2 (7.14) 4 (14.28) 1 (3.57) 2 (7.14) 3 (10.71) 1 (3.57) 4 (14.28)
0.61 ± 0.15 1 (9.09) 4 (36.36) 0 (0.00) 0 (0.00) 1 (9.09) 5 (45.45)§ 2 (18.18)
1.02 ± 0.05# 1 (2.50) 6 (15.00) 2 (5.00) 3(7.50) 1 (2.50) 3 (7.50) 1 (2.22)
0 0 0 0 0 0 0
0 0 0 0 0 0 0
3.43 ± 2.52 (1–9) 0.60 ± 0.07 1 (4.35) 6 (26.09) 2 (8.70) 1 (4.35) 7 (30.43) 2 (8.70) 8 (34.78)
⁎ Statistically significant in comparison with CIDP, GBS, MFS, MS and NC. # Statistically significant in comparison with CIDP, GBS and MFS. § Statistically significant in comparison with CIDP, MFS and MS.
patients. These findings indicate that TGF-β1 could be a potential biomarker for the evaluation of disease severity in GBS. Alteration of TGF-β1 concentrations in body fluids has been detected in neurological diseases such as amyotrophic lateral sclerosis (ALS) [25], AD [26,27] and GBS [28,29]. Plasma TGF-β1 concentrations are significantly higher in ALS patients, while a significant positive correlation between the TGF-β1 plasma concentration in patients with ALS and the duration of illness was also demonstrated [25]. Patients with AD display
higher concentrations of TGF-β1 in CSF than control subjects [27], whereas the TGF-β1 concentration in the plasma of AD is reduced [26]. In GBS, Ossege et al. found that serum concentrations of TGF-β1 were significantly increased at the nadir of disease course (acute phase) and recovery phase of GBS, whereas the number of TGF-β1 secreting cells was Increased in all phases of GBS [29]. Our study showed similar results to those of Ossege et al. Créange et al. reported that TGFβ1 expression was decreased in the plasma of GBS patients at the acute
Fig. 1. Serum levels of (A) TGF-β1, (B) IL-6, (C) VCAM-1 and (D) VEGF in patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain-Barré syndrome (GBS), Miller-Fisher syndrome (MFS), multiple sclerosis (MS), Alzheimer's disease (AD) and healthy controls (HC). *: Statistically significant in comparison with CIDP, MFS, MS, AD and NC, P b 0.05, analysis of variance and Bonferroni post hoc test.
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Table 2 The correlation between TGF-β1 and disease severity in patients with Guillain-Barré syndrome (GBS), Miller-Fisher syndrome (MFS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Disease (scale)
β coefficient (ng/ml)
P value⁎
GBS (Hughes' functional grading scale) MFS (Hughes' functional grading scale) CIDP (INCAT disability scale)
6.05 ± 1.26 1.83 ± 1.08 0.45 ± 0.53
b0.001 0.09 0.40
⁎ P value of generalized linear model (GLM) with adjustment of age and gender.
Fig. 2. (A–B) Relationship between serum TGF-β1 level and Hughes' functional grading scale in the patients with Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS). (C) Relationship between serum TGF-β1 level and INCAT disability scale in the patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).
phase and increased gradually back to the normal concentration at the recovery stage [28]. The blood samples showing increasing TGF-β1 in the study by Créange et al., were collected after treatment with plasmapheresis or intravenous immunoglobulin. It is worthy to note that our GBS patients demonstrating increased serum concentrations of TGF-β1 were treatment-naïve and treatments may alter the inflammatory responses, which may explain the discrepancy between our study results and those shown by Créange et al. Importantly, our data showed a positive correlation between serum concentrations of TGF-β1 and the degree of disability in GBS as well as an adequate AUC of the ROC curve analysis, which may strengthen the potential of serum TGF-β1 as a biomarker for GBS. The difference of serum concentrations of TGF-β1 between GBS and CIDP indicates diverse immunopathogenesis of these 2 peripheral inflammatory neuropathies. The exact mechanism underlying increased serum concentrations of TGF-β1 in GBS patients is still unclear. It has been shown that mRNA expression of TGF-β1 in leukocytes is significantly up-regulated during the disease course [29,30], which paralleled serum concentrations of TGFβ1 [29]. On the other hand, CSF concentrations of TGF-β1 concentrations were undetectable. Therefore we postulated that TGF-β1 in the serum of GBS patients is mainly produced by peripheral leukocytes. TGF-β1 is a multifunctional cytokine that regulates a broad diversity of physiological and pathological processes of many immune-cell types [31]. In T lymphocytes, TGF-β1 blocks their proliferation by inhibiting IL-2 production and down-regulating the expression of cyclin D2 and E, cyclin-dependent kinase 4, and c-MYC [32–35]. TGF-β1 may also suppress differentiation of T helper 1 lymphocytes by inhibiting TBX21 expression [36]. Furthermore, TGF-β1 prevents T cell activation-induced cell death via down-regulating c-MYC-mediated Fas ligand expression [33]. In an experimental model of spinal cord demyelination, upregulation of TGF-β1 was associated with remyelination [37]. The above evidence suggests that TGF-β1 is an anti-inflammatory factor. Therefore, the increased serum TGF-β1 concentrations in GBS patients probably represent a compensatory response to the inflammatory process. It has been shown that TGF-β1 may be highly expressed in cancer cells [38], while occasionally GBS was caused by paraneoplastic polyneuropathy [39]. Therefore, we measured a panel of paraneoplastic anti-neuronal antibodies. None of these patients demonstrated these paraneoplastic auto-antibodies. Previous studies showed that IL-6 was detectable in serum and CSF of a few patients with GBS and CIDP, whereas IL-6 concentrations were not quantified [40,41]. However, our quantification results showed that serum concentrations of IL-6 were similar in these 2 inflammatory neuropathies and when compared to other groups of diseases and controls. Increased serum concentrations of VCAM-1 have been specifically seen in GBS patients with antecedent cytomegalovirus infection, which however only accounts for 8% of GBS patients [42]. Consistent with our findings, unchanged serum concentrations of VEGF in GBS and CIDP patients were reported [43]. Our results are similar to the previous reports that serum concentrations of IL-6, VCAM-1 and VEGF in MS patients were not altered [44,45]. In our study, only 45% of patients with MFS had anti-GQ1b IgG, whereas other series described frequencies above 90% [46,47]. This discrepancy is probably due to immunoblot assay used to detect anti-
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ganglioside antibodies, which is not the expert-recommended enzymelinked immunosorbent assay in literature [48]. Nobile-Orazio et al. found higher serum VEGF concentrations in patients with GBS, CIDP and other immune-mediated neuropathies [49]. However, our results did not recapitulate these features. A larger case-control series will be needed to clarify the role of VEGF in immune-mediated neuropathies. In conclusion, we found that serum concentration of TGF-β1 was increased in GBS patients compared to patients with other neurological diseases, including CIDP, MFS, MS and AD, as well as control subjects. These findings implicate TGF-β1 may be involved in the compensatory response to the inflammatory process of GBS. Several molecular biomarkers have been found to be associated with the pathogenesis, development, or recovery of GBS [50]. Some of them, such as IL-37, IL-17, IL22, and TNF-α were down-regulated by IVIg treatment [8,9,51]. Therefore, further studies to compare the concentrations before and after treatment in the same group of GBS patients may consolidate the role of TGF-β1 in GBS patients. Given that the number of our patients is small, a larger, multi-center, and prospective study focusing on the correlation of serum concentrations of TGF-β1 with clinical or electrophysiological features will be mandatory. A standard protocol to measure the biomarkers should be established to avoid conflicting results by using different methods. Acknowledgments We thank all the patients and the staffs at the Department of Neurology, Chang Gung Memorial Hospital Linkou Medical Center for their valuable support of this study. This study was sponsored by Chang Gung Memorial Hospital (grants CMRPG3C1631, 3C1632, and 3C1633), Taipei, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References [1] B. van den Berg, C. Walgaard, J. Drenthen, C. Fokke, B.C. Jacobs, P.A. van Doorn, Guillain-Barré syndrome: pathogenesis, diagnosis, treatment and prognosis, Nat. Rev. Neurol. 10 (2014) 469–482. [2] Report from an Ad Hoc Subcommittee of the American Academy of Neurology AIDS Task Force, Research criteria for diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP), Neurology 41 (1991) 617–618. [3] G. Chavada, H.J. Willison, Autoantibodies in immune-mediated neuropathies, Curr. Opin. Neurol. 25 (2012) 550–555. [4] K. Kaida, T. Ariga, R.K. Yu, Antiganglioside antibodies and their pathophysiological effects on Guillain–Barré syndrome and related disorders—a review, Glycobiology 19 (2009) 676–692. [5] T. Jin, L.S. Hu, M. Chang, J. Wu, B. Winblad, J. Zhu, Proteomic identification of potential protein markers in cerebrospinal fluid of GBS patients, Eur. J. Neurol. 14 (2007) 563–568. [6] K.H. Chang, R.K. Lyu, M.Y. Tseng, et al., Elevated haptoglobin level of cerebrospinal fluid in Guillain-Barré syndrome revealed by proteomics analysis, Proteomics Clin. Appl. 1 (2007) 467–475. [7] H.L. Zhang, X.M. Zhang, X.J. Mao, et al., Altered cerebrospinal fluid index of prealbumin, fibrinogen, and haptoglobin in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy, Acta Neurol. Scand. 125 (2012) 129–135. [8] C. Li, P. Zhao, X. Sun, Y. Che, Y. Jiang, Elevated levels of cerebrospinal fluid and plasma interleukin-37 in patients with Guillain-Barré syndrome, Mediat. Inflamm. 2013 (2013) 639712. [9] S. Li, T. Jin, H.L. Zhang, et al., Circulating Th17, Th22, and Th1 cells are elevated in the Guillain-Barré syndrome and downregulated by IVIg treatments, Mediat. Inflamm. 2014 (2014) 740947. [10] S. Jander, G. Stoll, Interleukin-18 is induced in acute inflammatory demyelinating polyneuropathy, J. Neuroimmunol. 114 (2001) 253–258. [11] C.L. Koski, M.E. Sanders, P.T. Swoveland, et al., Activation of terminal components of complement in patients with Guillain-Barré syndrome and other demyelinating neuropathies, J. Clin. Invest. 80 (1987) 1492–1497. [12] K.H. Chang, T.J. Chuang, R.K. Lyu, et al., Identification of gene networks and pathways associated with Guillain-Barré syndrome, PLoS One 7 (2012), e29506. [13] J. Gaiottino, N. Norgren, R. Dobson, et al., Increased neurofilament light chain blood levels in neurodegenerative neurological diseases, PLoS One 8 (2013), e75091. [14] H. Maier, M. Schmidbauer, B. Pfausler, E. Schmutzhard, H. Budka, Central nervous system pathology in patients with the Guillain-Barré syndrome, Brain 120 (1997) 451–464.
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Increased serum concentrations of transforming growth factor-β1 (TGF-β1) in patients with Guillain-Barré syndrome Kuo-Hsuan Chang, Rong-Kuo Lyu, Yen-Shi Ro, Yi-Chun Chen, Long-Sun Ro, Hong-Shiu Chang, Ching-Chang Huang, Ming-Feng Liao, Yih-Ru Wu, Hong-Chou Kuo, Chun-Che Chu, Chiung-Mei Chen ⁎ Department of Neurology, Chang Gung Memorial Hospital-Linkou Medical Center, Chang Gung University College of Medicine, Taoyuan, Taiwan
a r t i c l e
i n f o
Article history: Received 12 April 2016 Received in revised form 19 July 2016 Accepted 19 July 2016 Available online 20 July 2016 Keywords: Guillain-Barré syndrome TGF-β1 Biomarker
a b s t r a c t Background: Guillain-Barré syndrome (GBS) is an acquired demyelinating peripheral neuropathy. It has shown that macrophage activation contribute to the pathogenesis of GBS. Therefore macrophage-mediated factors could be the potential markers for disease diagnosis and status of GBS. Methods: We measured serum concentrations of 4 macrophage-mediated factors, including interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), vascular cell adhesion protein 1 (VCAM-1) and vascular endothelial growth factor (VEGF), in 23 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), 28 GBS, 11 Miller-Fisher syndrome (MFS), 40 multiple sclerosis (MS), and 12 Alzheimer's disease (AD) patients, as well as 15 healthy controls. Results: Serum TGF-β1 concentration of GBS patients (35.94 ± 2.55 ng/ml) was significantly higher compared with CIDP (25.46 ± 1.40 ng/ml, P b 0.001), MFS (25.32 ± 2.31 ng/ml, P = 0.010), MS (21.35 ± 0.90 ng/ml, P b 0.001) and AD patients (22.92 ± 1.82 ng/ml, P b 0.001), as well as healthy controls (23.12 ± 1.67 ng/ml, P b 0.001). A positive correlation between serum TGF-β1 concentrations and Hughes' functional grading scales was observed in GBS patients. Serum concentrations of IL-6, VCAM-1 and VEGF were similar between the studied groups. Conclusion: The high serum concentrations of TGF-β1 and the correlation between serum TGF-β1 concentration and disease severity highlight the potential of TGF-β1 as a biomarker of GBS. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Guillain-Barré syndrome (GBS) is an acquired acute inflammatory peripheral neuropathies [1]. According to different pathophysiological and clinical features, GBS can be classified into several subtypes such as acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), and Miller-Fisher syndrome (MFS) [1]. GBS shares many symptoms and signs with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) particularly in the acute phase of disease. The time to nadir and the subsequent course of the disease
Abbreviations: AD, Alzheimer's disease; AIDP, acute inflammatory demyelinating polyneuropathy; AMAN, acute motor axonal neuropathy; AMSAN, acute motor and sensory axonal neuropathy; CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; CSF, cerebrospinal fluid; GBS, Guillain-Barré syndrome; IL-6, interleukin-6; INCAT disability scale, inflammatory neuropathy cause and treatment disability scale; MFS, Miller-Fisher syndrome; MS, multiple sclerosis; TGF-β1, transforming growth factor-β1; VCAM-1, vascular cell adhesion protein 1; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Department of Neurology, Chang Gung Memorial HospitalLinkou Medical Center, No.5, Fusing St., Gueishan Township, Taoyuan County 333, Taiwan. E-mail address: [email protected] (C.-M. Chen).
http://dx.doi.org/10.1016/j.cca.2016.07.013 0009-8981/© 2016 Elsevier B.V. All rights reserved.
are important clinical differences between these 2 diseases. GBS is a monophasic disease in which the time to reach nadir by definition is within 4 weeks [1], whereas the initial progressive phase lasts N 2 months will be considered as CIDP [2]. The diagnosis of GBS is rather based on a combination of clinical, electrophysiological features as well as analysis of the cerebrospinal fluid (CSF) at present [1]. However, the electrophysiological and CSF examinations fail to show the abnormalities at the early stage of GBS [1]. A biomarker that can be evaluated as an indicator of a pathological process or pharmacological response to a therapeutic intervention could assist in the clinical diagnosis, monitoring disease progression, and testing the efficacy of immunotherapy in GBS. The pathogenesis of GBS is thought to be immune-mediated [3]. This immune response, possibly triggered by antecedent infection [1], may generate antibodies that cross-react with gangliosides at myelin, resulting in slowness or blockade of nerve conduction as well as damage of axons by inflammatory infiltrates [4]. Given that inflammation in GBS could be driven by peripheral lymphocytes, discovery of GBS-specific inflammatory biomarkers in peripheral blood has been repeatedly attempted. So far, a list of GBS-associated biomarkers including haptoglobin [5,6], prealbumin [7], interferon-γ [8], tumor necrosis factor-α [8], interleukin (IL)-17 [9], IL-18 [10], IL-22 [9], IL-37 [8], soluble C5b-9
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complex complement [11], matrix metalloproteinase-9 [12] and neurofilaments [13] has been disclosed in blood or CSF samples. All of these results support the critical role of peripheral inflammation in the pathogenesis GBS. Pathologically, the inflammation of GBS may not be limited to peripheral nerves. Inflammatory infiltrates are also found in spinal cord of GBS patients [14]. The major part of inflammatory cells in GBS, activated macrophage [15], can secrete cytokines, such as IL-6 [16], transforming growth factor-β1 (TGF-β1) [16], vascular cell adhesion protein 1 (VCAM-1) [17], and vascular endothelial growth factor (VEGF) [16], which are usually associated with pro-inflammatory or anti-inflammatory responses. 2. Subjects and methods 2.1. Ethics statement This study was performed under a protocol approved by the institutional review boards of Chang Gung Memorial Hospital (ethical license No: 103-4555C) and all examinations were performed after obtaining written informed consents. 2.2. Sample collection Serum samples were collected from 23 CIDP (10 females, 13 males), 28 GBS (15 females, 13 males, 11 MFS (5 females, 6 males), 40 MS (28 females, 12 males), and 12 AD (7 females, 5 males) patients, as well as 15 healthy controls (7 females, 8 males). The diagnosis of GBS [18], CIDP [19], MFS [20], MS [21] and AD [22] were made according to respective diagnostic criteria. The neurological disability of the patients at the time of nadir was assessed using the Hughes' functional grading scale (for patients with GBS and MFS) [23] or Inflammatory Neuropathy Cause and Treatment (INCAT) disability scale (for patients with CIDP) [24] on the basis of the results of neurological examination and ambulatory ability assessment. All subjects were confirmed to have no systemic infection, chronic renal failure, cardiac or liver dysfunction, malignancies, or autoimmune diseases other than GBS, CIDP, MFS, MS and AD. In the patients with GBS, MFS and MS, serum and CSF samples were collected within 2 weeks after the onset or during acute relapses of diseases. All samples were obtained before treatment with plasmapheresis, or intravenous immunoglobulins. Sample analyses were blindly performed with respect to patients' diagnosis and the results of other tests from the same patient. Serum samples were kept sitting at 4 °C for 4 h and then centrifuged, aliquoted, frozen at − 80 °C, and stored until analysis. The albumin and immunoglobulin (IgG) concentrations in serum and CSF were measured by the Department of Clinical Pathology, Chang Gung Memorial Hospital. 2.3. Anti-ganglioside and anti-neuronal antigen antibodies assay Anti-ganglioside (anti-GD1a, anti-GD1b, anti-GM1, anti-GM2, antiGM3, anti-GQ1b and anti-GT1b) and anti-neuronal antigen (antiAMPHIPHYSIN, anti-CV2, anti-Hu, anti-Ma2, anti-RECOVERIN, anti-Ri, anti-SOX1, anti-TITIN and anti-Yo) IgG autoantibodies were measured by using immunoblot strip kit (Euroimmun AG) according to the manufacturer's instruction. 2.4. Enzyme-linked immunosorbent assays for quantification of targeted inflammatory markers Serum concentrations of IL-6, TGF-β1, VEGF and VCAM-1 were assessed using enzyme-linked immunosorbent assay (ELISA) (R & D Systems). Each assay was performed in duplicate according to the manufacturer's instruction.
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2.5. Statistical analysis The Statistical Program for Social Sciences (SPSS) was used to analyze all the statistics. Non-categorical variables were compared using the Student's t-test, 1-way analysis of variance with Bonferroni post hoc test. Generalized linear model with adjustment for age and gender was applied to evaluate the correlation between serum TGF-β1 concentrations and the Hughes' functional grading scale or INCAT disability scale at the time of sample collection. Each set of data was expressed as mean ± standard error. All P values were 2-tailed, and a P b 0.05 was considered significant. Receiver operating characteristic curve analysis was used to measure the ability of TGF-β1 concentration to predict the diagnosis of GBS. 3. Results The demographic data of all groups were displayed in Table 1. Not surprisingly, the age of the patients with AD (75.7 ± 1.63 y) was significantly higher than the other groups (P b 0.001). IgG index in the patient with MS (1.02 ± 0.05) was significant higher compared with those with CIDP (0.60 ± 0.07, P = 0.002), GBS (0.69 ± 0.05, P = 0.001) and MFS (0.61 ± 0.15, P = 0.026). A panel of serum anti-ganglionside IgG auto-antibodies including anti-GM1, anti-GM2, anti-GM3, anti-GD1a, anti-GD1b, anti-GT1b and anti-GQ1b were measured for these patients. The results showed a number of CIDP patients had anti-GT1b (34.78%), anti-GM3 (30.43%) and anti-GD1b (26.09%). These auto-antibodies were present in fewer patients with GBS compared with those with CIDP, although the differences did not reach the statistical significance. Anti-GQ1b, thought as a diagnostic marker for MFS, was significantly increased (45.45%) in patients with MFS when compared with the other groups (P = 0.008–0.048). Paraneoplastic anti-neuronal antibodies including anti-AMPHIPHYSIN, anti-CV2, anti-Hu, anti-Ma2, antiRECOVERIN, anti-Ri, anti-SOX1, anti-TITIN and anti-Yo were negative in all patients. Activation of macrophage plays an important role in the peripheral neuroinflammatory diseases. By examining four macrophage-mediated inflammatory factors, IL-6, TGF-β1, VCAM-1 and VEGF, in the patients with CIDP, GBS, MFS, MS, and AD, as well as healthy controls, we found serum concentrations of TGF-β1 were significantly increased in GBS patients (35.94 ± 2.55 ng/ml, Fig. 1A) compared to those with CIDP (25.46 ± 1.40 ng/ml, P b 0.001), MFS (25.32 ± 2.31 ng/ml, P = 0.010), MS (21.35 ± 0.90 ng/ml, P b 0.001) and AD (22.92 ± 1.82 ng/ ml, P b 0.001), as well as healthy controls (23.12 ± 1.67 ng/ml, P b 0.001). The area under ROC curve (AUC) for TGF-β1 was 0.78. None of the other three markers showed significant differences between the six groups (Fig. 1B–D). We analyzed the correlation between the serum concentrations of TGF-β1 and disease severity that was evaluated by scores of Hughes' functional grading scale (GBS and MFS) or INCAT disability scale (CIDP) at the time point of sample collection. The results showed a significant correlation between serum concentrations of TGF-β1 and Hughes' functional grading scale in the patients with GBS (β coefficient: 6.05 ± 1.26 ng/ml, P b 0.001, Fig. 2A and Table 2). On the other hand, such clinical correlation is absent in the patients with MFS and CIDP (Fig. 2B and C, Table 2). The CSF concentration of TGF-β1 was undetectable by the assay we used. 4. Discussion Given that a peripheral nervous tissue sample from GBS patients is practically difficult to access, biomarkers in blood should be more feasible as an indicator for the disease severity as well as testing potential therapeutic strategies. In the present study, we demonstrated that serum TGF-β1 concentration was higher in the GBS patients when compared with MFS, CIDP and AD patients, as well as controls. Furthermore, a significant correlation between serum TGF-β1 concentrations and Hughes' functional grading scale scores was demonstrated in GBS
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Table 1 Clinical characteristics of the patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain-Barré syndrome (GBS), Miller-Fisher syndrome (MFS), multiple sclerosis (MS), Alzheimer's disease (AD) and healthy controls (HC). Parameter
CIDP (n = 23)
GBS (n = 28)
MFS (n = 11)
MS (n = 40)
AD (n = 12)
HC (n = 15)
Gender (male/female) Age (y) (range) Hughes' functional grading scale (range) INCAT disability scale (range) IgG index Anti-GD1a IgG (%) Anti-GD1b IgG (%) Anti-GM1 IgG (%) Anti-GM2 IgG (%) Anti-GM3 IgG (%) Anti-GQ1b IgG (%) Anti-GT1b IgG (%)
13/10 50.0 ± 2.9 (22–80)
13/15 47.0 ± 3.4 (27–83) 2.21 ± 1.52 (1–5)
6/5 43.1 ± 4.8 (18–65) 1.82 ± 0.98 (1–3)
12/28 37.3 ± 1.8 (19–65)
5/7 75.7 ± 1.6⁎ (63–84)
8/7 47.0 ± 156 (27–84)
0.69 ± 0.05 2 (7.14) 4 (14.28) 1 (3.57) 2 (7.14) 3 (10.71) 1 (3.57) 4 (14.28)
0.61 ± 0.15 1 (9.09) 4 (36.36) 0 (0.00) 0 (0.00) 1 (9.09) 5 (45.45)§ 2 (18.18)
1.02 ± 0.05# 1 (2.50) 6 (15.00) 2 (5.00) 3(7.50) 1 (2.50) 3 (7.50) 1 (2.22)
0 0 0 0 0 0 0
0 0 0 0 0 0 0
3.43 ± 2.52 (1–9) 0.60 ± 0.07 1 (4.35) 6 (26.09) 2 (8.70) 1 (4.35) 7 (30.43) 2 (8.70) 8 (34.78)
⁎ Statistically significant in comparison with CIDP, GBS, MFS, MS and NC. # Statistically significant in comparison with CIDP, GBS and MFS. § Statistically significant in comparison with CIDP, MFS and MS.
patients. These findings indicate that TGF-β1 could be a potential biomarker for the evaluation of disease severity in GBS. Alteration of TGF-β1 concentrations in body fluids has been detected in neurological diseases such as amyotrophic lateral sclerosis (ALS) [25], AD [26,27] and GBS [28,29]. Plasma TGF-β1 concentrations are significantly higher in ALS patients, while a significant positive correlation between the TGF-β1 plasma concentration in patients with ALS and the duration of illness was also demonstrated [25]. Patients with AD display
higher concentrations of TGF-β1 in CSF than control subjects [27], whereas the TGF-β1 concentration in the plasma of AD is reduced [26]. In GBS, Ossege et al. found that serum concentrations of TGF-β1 were significantly increased at the nadir of disease course (acute phase) and recovery phase of GBS, whereas the number of TGF-β1 secreting cells was Increased in all phases of GBS [29]. Our study showed similar results to those of Ossege et al. Créange et al. reported that TGFβ1 expression was decreased in the plasma of GBS patients at the acute
Fig. 1. Serum levels of (A) TGF-β1, (B) IL-6, (C) VCAM-1 and (D) VEGF in patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain-Barré syndrome (GBS), Miller-Fisher syndrome (MFS), multiple sclerosis (MS), Alzheimer's disease (AD) and healthy controls (HC). *: Statistically significant in comparison with CIDP, MFS, MS, AD and NC, P b 0.05, analysis of variance and Bonferroni post hoc test.
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Table 2 The correlation between TGF-β1 and disease severity in patients with Guillain-Barré syndrome (GBS), Miller-Fisher syndrome (MFS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Disease (scale)
β coefficient (ng/ml)
P value⁎
GBS (Hughes' functional grading scale) MFS (Hughes' functional grading scale) CIDP (INCAT disability scale)
6.05 ± 1.26 1.83 ± 1.08 0.45 ± 0.53
b0.001 0.09 0.40
⁎ P value of generalized linear model (GLM) with adjustment of age and gender.
Fig. 2. (A–B) Relationship between serum TGF-β1 level and Hughes' functional grading scale in the patients with Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS). (C) Relationship between serum TGF-β1 level and INCAT disability scale in the patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).
phase and increased gradually back to the normal concentration at the recovery stage [28]. The blood samples showing increasing TGF-β1 in the study by Créange et al., were collected after treatment with plasmapheresis or intravenous immunoglobulin. It is worthy to note that our GBS patients demonstrating increased serum concentrations of TGF-β1 were treatment-naïve and treatments may alter the inflammatory responses, which may explain the discrepancy between our study results and those shown by Créange et al. Importantly, our data showed a positive correlation between serum concentrations of TGF-β1 and the degree of disability in GBS as well as an adequate AUC of the ROC curve analysis, which may strengthen the potential of serum TGF-β1 as a biomarker for GBS. The difference of serum concentrations of TGF-β1 between GBS and CIDP indicates diverse immunopathogenesis of these 2 peripheral inflammatory neuropathies. The exact mechanism underlying increased serum concentrations of TGF-β1 in GBS patients is still unclear. It has been shown that mRNA expression of TGF-β1 in leukocytes is significantly up-regulated during the disease course [29,30], which paralleled serum concentrations of TGFβ1 [29]. On the other hand, CSF concentrations of TGF-β1 concentrations were undetectable. Therefore we postulated that TGF-β1 in the serum of GBS patients is mainly produced by peripheral leukocytes. TGF-β1 is a multifunctional cytokine that regulates a broad diversity of physiological and pathological processes of many immune-cell types [31]. In T lymphocytes, TGF-β1 blocks their proliferation by inhibiting IL-2 production and down-regulating the expression of cyclin D2 and E, cyclin-dependent kinase 4, and c-MYC [32–35]. TGF-β1 may also suppress differentiation of T helper 1 lymphocytes by inhibiting TBX21 expression [36]. Furthermore, TGF-β1 prevents T cell activation-induced cell death via down-regulating c-MYC-mediated Fas ligand expression [33]. In an experimental model of spinal cord demyelination, upregulation of TGF-β1 was associated with remyelination [37]. The above evidence suggests that TGF-β1 is an anti-inflammatory factor. Therefore, the increased serum TGF-β1 concentrations in GBS patients probably represent a compensatory response to the inflammatory process. It has been shown that TGF-β1 may be highly expressed in cancer cells [38], while occasionally GBS was caused by paraneoplastic polyneuropathy [39]. Therefore, we measured a panel of paraneoplastic anti-neuronal antibodies. None of these patients demonstrated these paraneoplastic auto-antibodies. Previous studies showed that IL-6 was detectable in serum and CSF of a few patients with GBS and CIDP, whereas IL-6 concentrations were not quantified [40,41]. However, our quantification results showed that serum concentrations of IL-6 were similar in these 2 inflammatory neuropathies and when compared to other groups of diseases and controls. Increased serum concentrations of VCAM-1 have been specifically seen in GBS patients with antecedent cytomegalovirus infection, which however only accounts for 8% of GBS patients [42]. Consistent with our findings, unchanged serum concentrations of VEGF in GBS and CIDP patients were reported [43]. Our results are similar to the previous reports that serum concentrations of IL-6, VCAM-1 and VEGF in MS patients were not altered [44,45]. In our study, only 45% of patients with MFS had anti-GQ1b IgG, whereas other series described frequencies above 90% [46,47]. This discrepancy is probably due to immunoblot assay used to detect anti-
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ganglioside antibodies, which is not the expert-recommended enzymelinked immunosorbent assay in literature [48]. Nobile-Orazio et al. found higher serum VEGF concentrations in patients with GBS, CIDP and other immune-mediated neuropathies [49]. However, our results did not recapitulate these features. A larger case-control series will be needed to clarify the role of VEGF in immune-mediated neuropathies. In conclusion, we found that serum concentration of TGF-β1 was increased in GBS patients compared to patients with other neurological diseases, including CIDP, MFS, MS and AD, as well as control subjects. These findings implicate TGF-β1 may be involved in the compensatory response to the inflammatory process of GBS. Several molecular biomarkers have been found to be associated with the pathogenesis, development, or recovery of GBS [50]. Some of them, such as IL-37, IL-17, IL22, and TNF-α were down-regulated by IVIg treatment [8,9,51]. Therefore, further studies to compare the concentrations before and after treatment in the same group of GBS patients may consolidate the role of TGF-β1 in GBS patients. Given that the number of our patients is small, a larger, multi-center, and prospective study focusing on the correlation of serum concentrations of TGF-β1 with clinical or electrophysiological features will be mandatory. A standard protocol to measure the biomarkers should be established to avoid conflicting results by using different methods. Acknowledgments We thank all the patients and the staffs at the Department of Neurology, Chang Gung Memorial Hospital Linkou Medical Center for their valuable support of this study. This study was sponsored by Chang Gung Memorial Hospital (grants CMRPG3C1631, 3C1632, and 3C1633), Taipei, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References [1] B. van den Berg, C. Walgaard, J. Drenthen, C. Fokke, B.C. Jacobs, P.A. van Doorn, Guillain-Barré syndrome: pathogenesis, diagnosis, treatment and prognosis, Nat. Rev. Neurol. 10 (2014) 469–482. [2] Report from an Ad Hoc Subcommittee of the American Academy of Neurology AIDS Task Force, Research criteria for diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP), Neurology 41 (1991) 617–618. [3] G. Chavada, H.J. Willison, Autoantibodies in immune-mediated neuropathies, Curr. Opin. Neurol. 25 (2012) 550–555. [4] K. Kaida, T. Ariga, R.K. Yu, Antiganglioside antibodies and their pathophysiological effects on Guillain–Barré syndrome and related disorders—a review, Glycobiology 19 (2009) 676–692. [5] T. Jin, L.S. Hu, M. Chang, J. Wu, B. Winblad, J. Zhu, Proteomic identification of potential protein markers in cerebrospinal fluid of GBS patients, Eur. J. Neurol. 14 (2007) 563–568. [6] K.H. Chang, R.K. Lyu, M.Y. Tseng, et al., Elevated haptoglobin level of cerebrospinal fluid in Guillain-Barré syndrome revealed by proteomics analysis, Proteomics Clin. Appl. 1 (2007) 467–475. [7] H.L. Zhang, X.M. Zhang, X.J. Mao, et al., Altered cerebrospinal fluid index of prealbumin, fibrinogen, and haptoglobin in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy, Acta Neurol. Scand. 125 (2012) 129–135. [8] C. Li, P. Zhao, X. Sun, Y. Che, Y. Jiang, Elevated levels of cerebrospinal fluid and plasma interleukin-37 in patients with Guillain-Barré syndrome, Mediat. Inflamm. 2013 (2013) 639712. [9] S. Li, T. Jin, H.L. Zhang, et al., Circulating Th17, Th22, and Th1 cells are elevated in the Guillain-Barré syndrome and downregulated by IVIg treatments, Mediat. Inflamm. 2014 (2014) 740947. [10] S. Jander, G. Stoll, Interleukin-18 is induced in acute inflammatory demyelinating polyneuropathy, J. Neuroimmunol. 114 (2001) 253–258. [11] C.L. Koski, M.E. Sanders, P.T. Swoveland, et al., Activation of terminal components of complement in patients with Guillain-Barré syndrome and other demyelinating neuropathies, J. Clin. Invest. 80 (1987) 1492–1497. [12] K.H. Chang, T.J. Chuang, R.K. Lyu, et al., Identification of gene networks and pathways associated with Guillain-Barré syndrome, PLoS One 7 (2012), e29506. [13] J. Gaiottino, N. Norgren, R. Dobson, et al., Increased neurofilament light chain blood levels in neurodegenerative neurological diseases, PLoS One 8 (2013), e75091. [14] H. Maier, M. Schmidbauer, B. Pfausler, E. Schmutzhard, H. Budka, Central nervous system pathology in patients with the Guillain-Barré syndrome, Brain 120 (1997) 451–464.
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