Accepted Article
Received Date : 25-Mar-2014 Revised Date : 26-Jun-2014 Accepted Date : 20-Aug-2014 Article type
: Research Article
Soluble CD163 levels are elevated in cerebrospinal fluid and serum in people with Type 2 diabetes mellitus and are associated with impaired peripheral nerve function
M. Kallestrup1, H. J. Møller2, H. Tankisi3 and H Andersen1
1
Department
of
Neurology,
2
Department
of
Clinical
Biochemistry,
3
Department
of
Neurophysiology, Aarhus University Hospital, Aarhus, Denmark
Accepted Correspondence to: Mia-Maiken Kallestrup. E-mail: [email protected]
What's new? •
The present study found higher concentrations of the macrophage biomarker soluble CD163 in the cerebrospinal fluid and serum of people with Type 2 diabetes in comparison with people without diabetes.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/dme.12568 This article is protected by copyright. All rights reserved.
Accepted Article
•
•
Cerebrospinal fluid soluble CD163 level was higher in people with Type 2 diabetes and there was a trend for a higher level of soluble CD163 in cerebrospinal fluid and serum in people with neuropathy compared with those without neuropathy. Soluble CD163 was positively associated with impaired peripheral nerve conduction. The study indicates that inflammation may play a role in the development of impaired nerve function in people with Type 2 diabetes.
Abstract Aims To measure soluble CD163 levels in the cerebrospinal fluid and serum of people with Type 2 diabetes, with and without polyneuropathy, and to relate the findings to peripheral nerve function. Methods A total of 22 people with Type 2 diabetes and 12 control subjects without diabetes were included in this case–control study. Participants with diabetes were divided into those with neuropathy (n=8) and those without neuropathy (n=14) based on clinical examination, vibratory perception thresholds and nerve conduction studies. Serum and cerebrospinal fluid soluble CD163 levels were analysed using an enzyme-linked immunosorbent assay. Results Soluble CD163 levels were significantly higher in the cerebrospinal fluid and serum of the participants with Type 1 diabetes compared with the control participants [cerebrospinal fluid: median (range) 107 (70–190) vs 84 (54–115) µg/l, P < 0.01 and serum: 2305 (920–7060) vs 1420 (780–2740) µg/l, P < 0.01). Cerebrospinal fluid soluble CD163 was positively related to impaired
peripheral nerve conduction (nerve conduction study rank score: r = 0.42; P = 0.0497) and there was a trend for higher levels of soluble CD163 in the cerebrospinal fluid and serum in participants with neuropathy than in those without neuropathy [cerebrospinal fluid: median (range) 131 (86– 173) vs 101 (70–190) µg/l, P = 0.08 and serum: 3725 (920–7060) vs 2220 (1130–4780), P = 0.06).
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Conclusions Cerebrospinal fluid soluble CD163 level is associated with impaired peripheral nerve function. Higher levels of soluble CD163 in people with diabetic polyneuropathy suggest that inflammation plays a role in the development of neural impairment. The relationship between cerebrospinal fluid soluble CD163 level and peripheral nerve conduction indicates that soluble CD163 may be a potential biomarker for the severity of diabetic polyneuropathy.
Introduction Polyneuropathy is a common complication of both Type 1 and Type 2 diabetes mellitus and remains a major health problem worldwide, with a substantial risk of morbidity [1]. Nevertheless, the pathogenic factors underlying the development of diabetic polyneuropathy remain unclear. There is a growing body of evidence that inflammatory mediators originating from macrophages and other innate immune cells may be involved in the pathogenesis of diabetic polyneuropathy [2– 5]. Former studies indicate an association between subclinical inflammation and diabetic polyneuropathy in people with Type 2 diabetes [2,6]. Soluble CD163 is a macrophage-derived lowgrade inflammation marker reflecting macrophage activation. It is shed from the surface of CD163+ cells as a result of inflammatory stimuli; CD163 itself is an endocytic haptoglobin–haemoglobin receptor expressed on macrophages and monocytes [7]. The precise role of soluble CD163 is unknown, but soluble CD163 levels are elevated in many inflammatory diseases that involve macrophage activation, including low-grade inflammatory states such as Type 2 diabetes [8,9]. Soluble CD163 is strongly associated with later development of Type 2 diabetes in both lean and obese subjects, possibly as a result of macrophage infiltration of adipose tissue and the liver [10]. Type 2 diabetes is a systemic inflammatory disease in which elevated macrophage activation is found in the liver and adipose tissue [11]. Macrophages may also infiltrate nervous tissue and
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locally produce cytokines, resulting in endothelial and nerve fibre damage [12]; however, information on macrophage activation in nervous tissues in people with Type 2 diabetes and diabetic polyneuropathy is sparse. It would therefore be of particular interest to evaluate the degree of ongoing inflammation in the peripheral or central nervous system in people with diabetes and diabetic polyneuropathy. We hypothesized that activated macrophages take part in nerve degeneration in people with diabetes and our aim was to determine the concentration of soluble CD163 in CSF (cerebrospinal fluid) and blood in people with Type 2 diabetes, with and without diabetic polyneuropathy, as well as in control subjects without diabetes and to relate these findings to peripheral nerve function.
Subjects and methods This case–control study included people with Type 2 diabetes, with and without diabetic polyneuropathy (n=8 and n=14, respectively), and control subjects without diabetes (n=12). The study participants were consecutively recruited from outpatient clinics at the Department of Neurology and the Department of Medical Endocrinology at Aarhus University Hospital, Denmark, between November 2012 and August 2013. Participants with diabetes were diagnosed with diabetic polyneuropathy according to Dyck's minimal criteria [13]. Those with biochemical or neurophysiological evidence of other potential causes for the development of polyneuropathy were excluded. The control group consisted of people without diabetes with neurological symptoms leading to a lumbar puncture as part of the diagnostic evaluation. Exclusion criteria were diabetes, symptoms or signs of polyneuropathy or biochemical signs of inflammation or infection in the cerebrospinal fluid. Potential control subjects underwent an interview designed to ensure that none of the control group had symptoms of polyneuropathy, including diabetic polyneuropathy. All control participants
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had only mild neurological symptoms and, after diagnostic evaluation, none of them had a neurological disorder or showed signs of polyneuropathy. In all control participants included, the final diagnosis did not imitate polyneuropathy, including diabetic polyneuropathy. Furthermore, the final diagnoses of the control participants were believed to have a small, if any, impact on peripheral nerve function. Control participants were diagnosed with Bell’s palsy (n=9), benign muscle fasciculations (n=2) and idiopathic paresthesia (n=1). All cerebrospinal fluid samples were analysed with biochemical tests and none of the control participants displayed cerebrospinal fluid leukocytosis. Serum HbA1c levels were measured in all control participants to exclude those who
had diabetes. All participants gave informed consent to the study, which was approved by the local Ethics Committee and the Danish Data Protection Agency and performed in accordance with the Helsinki Declaration.
Diagnosis and staging of diabetic polyneuropathy Symptoms of peripheral neuropathy were assessed according to the neuropathy symptom score, which includes sensory, motor and autonomic symptoms of neuropathy. Participants with diabetes were evaluated with a standardized neurological examination performed by a trained neurologist (H.A.) resulting in a neuropathy impairment score, which is a combined score from the evaluation of muscle weakness, tendon reflexes and sensation. A high neuropathy impairment score or neuropathy symptom score reflects more severely impaired nerve function [14,15]. All participants with diabetes underwent nerve conduction studies with transcutaneous stimulation, using standardized recording procedures and electromyography equipment (Keypoint Medtronic, Skovlunde, Denmark). Skin temperatures were held above 32°C. Sensory nerve conduction velocity and compound sensory action potential amplitude were the variables evaluated for sensory nerve
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conduction studies, which were recorded in the dominant arm median nerve and bilateral sural nerves with antidromic stimulation. Motor nerve conduction velocity, distal motor latency, compound motor action potential amplitude and F-wave latency were the variables evaluated for motor nerve conduction studies recorded in the median nerve of the dominant arm and bilaterally in the tibial and peroneal nerves. Participants with median nerve impairment underwent additional sensory and motor nerve conduction studies of the dominant ulnar nerve to exclude carpal tunnel syndrome. Results within the range of mean ± two standard deviations compared with standard reference material were considered normal. Vibration perception thresholds were determined in all participants with diabetes using a computerbased sensory evaluation system (CASE IV, WR, Medical Electronics, Stillwater, MN, USA). Skin temperatures were held between 30 and 35 °C and sensory testing was performed at the dorsal site of the non-dominant first toe and dominant index finger. Diabetic polyneuropathy was diagnosed in participants with diabetes if two or more of the nerve conduction variables or vibration perception thresholds were abnormal: a neuropathy symptom score > 1; a neuropathy impairment score ≥ 2; a vibration perception threshold at the index finger and great toe ≥98th percentile; and abnormal motor or sensory nerve conduction velocity or abnormal compound motor or sensory action potential amplitude in at least two of four nerves: the dominant median nerve (motor and sensory), the non-dominant peroneal nerve or the non-dominant sural nerve [13]. For all participants with diabetes a nerve conduction study rank score was calculated. A high nerve conduction study rank score reflects more severely impaired peripheral nerve function. It was calculated as the sum of individual rankings of z-scores from the following nerve conduction variables: compound action potential amplitude and nerve conduction velocity of dominant median motor and sensory nerves; bilateral peroneal and tibial motor nerves; and bilateral sural sensory
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nerves. A neuropathy rank sum score was calculated for all participants with diabetes in order to rank the participants according to peripheral nerve function. The neuropathy rank sum score for each participant was calculated by adding the individual rank scores for neuropathy symptom score, neuropathy impairment score, nerve conduction study rank score and vibration perception threshold; a high neuropathy rank sum score reflected more impaired nerve function.
Measurement of soluble CD163 in cerebrospinal fluid and serum Blood and cerebrospinal fluid concentrations of soluble CD163 were determined in duplicates by use of an in-house sandwich enzyme-linked immunosorbent assay, essentially as described
previously [16]. In each run, we co-analysed control samples and the serum calibrator with concentrations traceable to purified CD163. Serum was measured on a BEP-2000 enzyme-linked immunosorbent assay-analyzer (Dade Behring, Marburg, Germany) diluted 1:101, and cerebrospinal fluid 1:3 before analyses. The inter-assay imprecision over the latest runs (n=159) was a 5.1% coefficient of variation at concentrations of 1.91 mg/l, and a 7.6% coefficient of variation at 3.83 mg/l. The limit of detection was 6.25 µg/l.
Clinical and standard biochemical measurements Standard biochemical measurements were performed by accredited methods at the Department of Clinical Biochemistry; serum HbA1c concentrations were measured in all participants. Furthermore,
to exclude competing causes to polyneuropathy, the following laboratory tests were performed in participants with diabetes: kidney, thyroid and liver function; immunoglobulin; M-component; and Wassermann reaction test. Cerebrospinal fluid protein concentration was measured in all study participants. Blood lipid concentrations were obtained from patient charts, but this information was missing in two control participants. Information regarding weight, height, gender, age and duration
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of diabetes (when applicable) were obtained for all study participants using patient interviews. In all participants with diabetes and 75% of the control participants, blood pressure was determined. Information on the use of anti-hypertensive, lipid-lowering and antiglycaemic drugs was obtained from patient charts.
Statistical analysis Data were analysed using STATA/IC_12 (StataCorp LP, TX, USA). For non-parametric data a
Kruskal–Wallis test was applied to address independence between groups. For parametric data Student's t-test was used to test for differences between two groups and for non-parametric data the
Mann–Whitney U-test was applied. To evaluate the statistical association between two variables, Spearman’s rank correlation was used. Two-tailed P-values < 0.05 were considered significant and
descriptive data are presented as median (range) values. A 30% difference in cerebrospinal fluid soluble CD163 concentration and a 50% difference in serum soluble CD163 concentration were expected between participants with diabetes and control participants. Based on previous studies of soluble CD163 concentration in cerebrospinal fluid in symptomatic control subjects (M. Stilund, unpublished data) and in serum in healthy control subjects [8], sample size calculations resulted in optimum group sizes of 10 participants to detect the difference in cerebrospinal fluid and 20 participants to detect the difference in serum (0.05 twosided significance level and 90% power).
Results Demographic characteristics of the study population Participant demographics and clinical information are shown in Table 1. The majority of the study population was male (79%). Participants with diabetes and control participants were similar with
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regard to age (P = 0.08), cerebrospinal fluid protein concentration (P = 0.09) and systolic blood
pressure (P = 0.73), while the two groups differed with regard to BMI (P = 0.01), serum HbA1c concentration (P < 0.01), serum total cholesterol concentration (P < 0.01) and number of
participants receiving anti-hypertensive or lipid-lowering treatment (P= 0.02 and P = 0.03,
respectively). Participants with diabetes, with and without polyneuropathy, were similar with regard to gender (P = 0.10), age (P = 0.81), BMI (P = 0.25), serum HbA1c and total cholesterol concentration (P = 0.16 and P = 0.30, respectively), systolic blood pressure (P= 0.06), diabetes duration (P = 0.84) and
number of participants receiving anti-hypertensive and lipid-lowering treatment (P = 0.11 and P =
0.43, respectively). Participants with diabetes and polyneuropathy had a higher cerebrospinal fluid protein concentration than participants with diabetes without diabetic polyneuropathy (P < 0.01). Notably, more participants without neuropathy than with neuropathy received insulin treatment (64 vs 13%; P = 0.02). As expected, the two groups differed in clinical neuropathy scores (neuropathy symptom score: P < 0.01; neuropathy impairment score: P < 0.01).
Comparison of cerebrospinal fluid and blood soluble CD163 levels and their association with peripheral nerve function Cerebrospinal fluid and serum soluble CD163 concentrations were higher in participants with diabetes than in control participants (Table 2). There was a trend towards higher levels of cerebrospinal fluid and serum soluble CD163 in participants with diabetes and polyneuropathy than in participants with diabetes without polyneuropathy [cerebrospinal fluid: median (range) 131 (86– 173) vs 101 (70–190) µg/l, P = 0.08 and serum: 3725 (920–7060) vs 2220 (1130–4780) µg/l, P = 0.06 (Figure 1)].
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When comparing participants with diabetes receiving s.c. administered insulin treatment with participants treated with oral antidiabetic drugs only, there was no difference in the concentration of soluble CD163 in cerebrospinal fluid [median (range): 109 (84–190) vs 105.5 (70–173) µg/l; P = 0.79] or serum [median (range): 2305 (1130–5000) vs 2480 (920–7060) µg/l, P = 0.90,
respectively]. Neurogenic pain was present in four out of eight participants with diabetic polyneuropathy. There was no difference in cerebrospinal fluid or serum soluble CD163 when comparing participants with diabetes and diabetic polyneuropathy with or without pain [cerebrospinal fluid: median (range) 131 (103–145) vs 129.5 (86–173) µg/l, P = 0.77 and serum: median (range) 4555 (1300–7060) vs 2825 (920–4010) µg/l, P = 0.15, respectively].
In analysis of all study participants, cerebrospinal fluid soluble CD163 showed a significant correlation with age, serum HbA1c and total cholesterol concentrations as well as with cerebrospinal
fluid protein levels (Table 3a). Serum soluble CD163 was associated with BMI and serum HbA1c and total cholesterol concentrations. In participants with diabetes cerebrospinal fluid soluble CD163 correlated to cerebrospinal fluid protein and nerve conduction study rank score (Fig. 2) but was unrelated to neuropathy rank sum score or neuropathy symptom score. There was a trend towards a positive correlation between cerebrospinal fluid soluble CD163 and neuropathy impairment score (P = 0.06; Table 3b). There
was no significant difference in cerebrospinal fluid soluble CD163–protein ratio between diabetic participants with and without polyneuropathy [median (range) score 207 (155–308) vs 263 (145– 363) P = 0.09].
Serum soluble CD163 did not correlate to cerebrospinal fluid protein or any measure of peripheral nerve function including nerve conduction study rank score, neuropathy rank sum score, neuropathy impairment score or neuropathy symptom score .
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In participants with diabetes, cerebrospinal fluid protein correlated with neuropathy symptom score (r = 0.51; P = 0.02), neuropathy impairment score (r = 0.67; P < 0.01), nerve conduction study rank score (r = 0.44; P = 0.04) and neuropathy rank sum score (r = 0.63; P < 0.01) but not with age (r = 0.09; P = 0.69).
Discussion To our knowledge, this is the first study of soluble CD163 in both cerebrospinal fluid and serum in people with diabetes and furthermore the first study to assess the relationship between soluble CD163 and neuropathy in diabetes. The findings lend support to the hypothesis that inflammation plays a role in the development of diabetic polyneuropathy. We found a higher concentration of soluble CD163 in cerebrospinal fluid and serum in participants with Type 2 diabetes compared with control participants. Moreover, there was a trend for higher levels of soluble CD163 in cerebrospinal fluid and serum in participants with diabetic polyneuropathy than in participants without it. Cerebrospinal fluid soluble CD163 was related to a marker of peripheral nerve conduction (nerve conduction study rank score: r = 0.42; P = 0.0497) and a trend towards a significant correlation to clinical signs of peripheral nerve impairment (neuropathy impairment score: r = 0.41; P = 0.06) was found.
The increase in cerebrospinal fluid soluble CD163 may be part of an inflammatory response and hence activation of peripheral nerve CD163+ macrophages in people with Type 2 diabetes and polyneuropathy. CD163+ macrophages and microglia are present in the central and peripheral nervous system and are believed to take part in immune-to-brain signalling and anti-inflammatory mechanisms [11]. Elevated levels of inflammatory markers in cerebrospinal fluid, such as tumour necrosis factor-α, may affect the central and peripheral nervous system and hence participate in the
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development of nervous complications in diabetes. Macrophage infiltration and expression of proinflammatory cytokines in the sciatic nerve has been reported in early phases of experimental diabetic polyneuropathy [17] as well as inflammatory cell infiltrations and immunological lesions in sural nerve biopsies in people with Type 1 and 2 diabetes [18]. Systemic inflammation, as seen in Type 2 diabetes, induces cytokine synthesis within the central nervous system [11] and in experimental Type 1 diabetes the presence of brain inflammation is suggested [19]. Soluble CD163 is transported passively across the blood–brain barrier and is also produced intrathecally [20]. The cerebrospinal fluid protein level, a marker of blood–brain barrier permeability, was higher in participants with diabetic polyneuropathy than in those without. Moreover, cerebrospinal fluid soluble CD163 concentration was positively associated with cerebrospinal fluid protein level in participants with diabetes. This may indicate that degeneration of the blood–brain barrier contributes to elevated cerebrospinal fluid soluble CD163 concentration; however, there was no statistically significant difference in cerebrospinal fluid soluble CD163– protein ratio between participants with or without diabetic polyneuropathy. It remains unclear to what extent the increased blood–brain barrier permeability adds to the increased concentration of cerebrospinal fluid soluble CD163 in participants with diabetic polyneuropathy compared with those without. Interestingly, the cerebrospinal fluid protein concentration correlated very closely to all markers of peripheral nerve function (neuropathy impairment score, neuropathy symptom score, nerve conduction study rank score and neuropathy rank sum score), which suggests that blood– brain barrier degeneration, which is accelerated in people with diabetes, plays a role in the development of diabetic polyneuropathy or alternatively reflects the pathogenetic process in diabetic polyneuropathy. Serum soluble CD163 was higher in participants with Type 2 diabetes than in control participants (Table 2) which is in accordance with former findings showing increased serum cytokine levels and
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hence subclinical inflammation in people with Type 2 diabetes [2,21–23]. Chronic hyperglycaemia activates macrophages [12] and may lead to elevated soluble CD163 levels. Adipose tissue is an important source of inflammatory factors in people with Type 2 diabetes [24] and a relationship between serum soluble CD163 and obesity in people with and without Type 2 diabetes has been shown [22]. In the present study, serum soluble CD163 was significantly associated with BMI (r = 0.4051; P = 0.0175). Nevertheless, the group of participants without diabetic polyneuropathy had the highest BMI, so disparity in BMI cannot explain the association between soluble CD163 and impaired nervous function.
Cerebrospinal fluid soluble CD163 levels correlated with the nerve conduction study rank score, suggesting an association between impaired peripheral nerve conduction and the inflammatory response in the peripheral and central nervous system. Inflammation may play a role in the development of diabetic polyneuropathy and soluble CD163 may be a biomarker reflecting the severity of diabetic polyneuropathy. Inflammatory markers correlate with non-inflammatory polyneuropathy severity [25] and inhibition of tumour necrosis factor-α, a pro-inflammatory mediator, has been shown to improve nerve conduction velocities and density of myelin nerve fibres in experimental diabetic polyneuropathy [26]. Hyperglycaemia-induced metabolic changes lead to dysregulation of cytokine control and this may lead to degeneration and reduced regenerative abilities in peripheral nerves [27,28]. Hyperglycaemia is believed to induce neurotoxicity via a series of pathways [29]; hence the hyperglycaemia itself may explain some degree of nerve degeneration seen in the present study. There is evidence to suggest a link between inflammation and painful diabetic polyneuropathy [3,30]. The present study offers no support to this notion; however, the number of participants
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included is too small to allow any conclusion about the impact of soluble CD163 on the development of pain. The main limitation of the present study is its small size and low number of study participants, which warrants larger studies in future; however, as a hypothesis-generating study, the findings add to the knowledge of the role of inflammation in diabetic polyneuropathy and, in future studies, the mechanisms should be studied in more detail. The control participants were chosen from a group of patients with mild neurological symptoms and this may have affected the results. Because of the limited number of participants we were unable to evaluate participants with asymptomatic diabetic polyneuropathy, but it would have been very interesting to evaluate if this had any effect on the soluble CD163 concentrations in cerebrospinal fluid or serum. Furthermore, because we focused on large fibre function only, the present study allows no conclusion as to whether soluble CD163 has any role in small fibre neuropathy. It is noteworthy that cerebrospinal fluid soluble CD163 only correlates with one marker of peripheral nerve impairment (nerve conduction study rank score); however, nerve conduction abnormalities may more closely reflect the severity of polyneuropathy than clinical examinations (neuropathy impairment score) or presence of neuropathy symptoms (neuropathy symptom score ).
In conclusion, in the present study we found higher levels of soluble CD163 in cerebrospinal fluid and serum in participants with Type 2 diabetes than in control participants. There was a trend for a higher level in neuropathic participants and cerebrospinal fluid soluble CD163 was associated with impaired peripheral nerve conduction. This is, to our knowledge, the first report of an association between soluble CD163 and a late diabetic complication. Future studies are needed to explore whether the innate immune system is activated in the central or peripheral nervous system in people with diabetes and, if so, how this may be related to peripheral nerve function.
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Funding sources The Department of Clinical Medicine, Aarhus University, Denmark, the BEVICA Foundation and the Danish council for strategic research (TRAIN10-092797) are acknowledged for funding of salary and biochemical analysis.
Competing interests H.J.M. has received royalties from IQ-products, The Netherlands. All other authors have no competing interests to declare.
Acknowledgements We thank our laboratory technician, Kirsten Bank Petersen, for excellent technical assistance.
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Neuropathy Study: design, criteria for types of neuropathy, selection bias, and reproducibility of neuropathic tests. Neurology 1991; 41: 799–807.
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14. Dyck P. Quantitating severity of neuropathy. In: Dyck P, Thomas P, Griffin J, Low P, Poduslo J, eds. Peripheral neuropathy. Philadelphia: WB Saunders, 1993. p. 686–97.
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FIGURE 1 The concentration of sCD163 in patients with Type 2 diabetes with and without neuropathy and
in control participants in (a) cerebrospinal fluid and b) serum. The three groups differed significantly with regard to soluble CD163 in cerebrospinal fluid (P
Received Date : 25-Mar-2014 Revised Date : 26-Jun-2014 Accepted Date : 20-Aug-2014 Article type
: Research Article
Soluble CD163 levels are elevated in cerebrospinal fluid and serum in people with Type 2 diabetes mellitus and are associated with impaired peripheral nerve function
M. Kallestrup1, H. J. Møller2, H. Tankisi3 and H Andersen1
1
Department
of
Neurology,
2
Department
of
Clinical
Biochemistry,
3
Department
of
Neurophysiology, Aarhus University Hospital, Aarhus, Denmark
Accepted Correspondence to: Mia-Maiken Kallestrup. E-mail: [email protected]
What's new? •
The present study found higher concentrations of the macrophage biomarker soluble CD163 in the cerebrospinal fluid and serum of people with Type 2 diabetes in comparison with people without diabetes.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/dme.12568 This article is protected by copyright. All rights reserved.
Accepted Article
•
•
Cerebrospinal fluid soluble CD163 level was higher in people with Type 2 diabetes and there was a trend for a higher level of soluble CD163 in cerebrospinal fluid and serum in people with neuropathy compared with those without neuropathy. Soluble CD163 was positively associated with impaired peripheral nerve conduction. The study indicates that inflammation may play a role in the development of impaired nerve function in people with Type 2 diabetes.
Abstract Aims To measure soluble CD163 levels in the cerebrospinal fluid and serum of people with Type 2 diabetes, with and without polyneuropathy, and to relate the findings to peripheral nerve function. Methods A total of 22 people with Type 2 diabetes and 12 control subjects without diabetes were included in this case–control study. Participants with diabetes were divided into those with neuropathy (n=8) and those without neuropathy (n=14) based on clinical examination, vibratory perception thresholds and nerve conduction studies. Serum and cerebrospinal fluid soluble CD163 levels were analysed using an enzyme-linked immunosorbent assay. Results Soluble CD163 levels were significantly higher in the cerebrospinal fluid and serum of the participants with Type 1 diabetes compared with the control participants [cerebrospinal fluid: median (range) 107 (70–190) vs 84 (54–115) µg/l, P < 0.01 and serum: 2305 (920–7060) vs 1420 (780–2740) µg/l, P < 0.01). Cerebrospinal fluid soluble CD163 was positively related to impaired
peripheral nerve conduction (nerve conduction study rank score: r = 0.42; P = 0.0497) and there was a trend for higher levels of soluble CD163 in the cerebrospinal fluid and serum in participants with neuropathy than in those without neuropathy [cerebrospinal fluid: median (range) 131 (86– 173) vs 101 (70–190) µg/l, P = 0.08 and serum: 3725 (920–7060) vs 2220 (1130–4780), P = 0.06).
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Conclusions Cerebrospinal fluid soluble CD163 level is associated with impaired peripheral nerve function. Higher levels of soluble CD163 in people with diabetic polyneuropathy suggest that inflammation plays a role in the development of neural impairment. The relationship between cerebrospinal fluid soluble CD163 level and peripheral nerve conduction indicates that soluble CD163 may be a potential biomarker for the severity of diabetic polyneuropathy.
Introduction Polyneuropathy is a common complication of both Type 1 and Type 2 diabetes mellitus and remains a major health problem worldwide, with a substantial risk of morbidity [1]. Nevertheless, the pathogenic factors underlying the development of diabetic polyneuropathy remain unclear. There is a growing body of evidence that inflammatory mediators originating from macrophages and other innate immune cells may be involved in the pathogenesis of diabetic polyneuropathy [2– 5]. Former studies indicate an association between subclinical inflammation and diabetic polyneuropathy in people with Type 2 diabetes [2,6]. Soluble CD163 is a macrophage-derived lowgrade inflammation marker reflecting macrophage activation. It is shed from the surface of CD163+ cells as a result of inflammatory stimuli; CD163 itself is an endocytic haptoglobin–haemoglobin receptor expressed on macrophages and monocytes [7]. The precise role of soluble CD163 is unknown, but soluble CD163 levels are elevated in many inflammatory diseases that involve macrophage activation, including low-grade inflammatory states such as Type 2 diabetes [8,9]. Soluble CD163 is strongly associated with later development of Type 2 diabetes in both lean and obese subjects, possibly as a result of macrophage infiltration of adipose tissue and the liver [10]. Type 2 diabetes is a systemic inflammatory disease in which elevated macrophage activation is found in the liver and adipose tissue [11]. Macrophages may also infiltrate nervous tissue and
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locally produce cytokines, resulting in endothelial and nerve fibre damage [12]; however, information on macrophage activation in nervous tissues in people with Type 2 diabetes and diabetic polyneuropathy is sparse. It would therefore be of particular interest to evaluate the degree of ongoing inflammation in the peripheral or central nervous system in people with diabetes and diabetic polyneuropathy. We hypothesized that activated macrophages take part in nerve degeneration in people with diabetes and our aim was to determine the concentration of soluble CD163 in CSF (cerebrospinal fluid) and blood in people with Type 2 diabetes, with and without diabetic polyneuropathy, as well as in control subjects without diabetes and to relate these findings to peripheral nerve function.
Subjects and methods This case–control study included people with Type 2 diabetes, with and without diabetic polyneuropathy (n=8 and n=14, respectively), and control subjects without diabetes (n=12). The study participants were consecutively recruited from outpatient clinics at the Department of Neurology and the Department of Medical Endocrinology at Aarhus University Hospital, Denmark, between November 2012 and August 2013. Participants with diabetes were diagnosed with diabetic polyneuropathy according to Dyck's minimal criteria [13]. Those with biochemical or neurophysiological evidence of other potential causes for the development of polyneuropathy were excluded. The control group consisted of people without diabetes with neurological symptoms leading to a lumbar puncture as part of the diagnostic evaluation. Exclusion criteria were diabetes, symptoms or signs of polyneuropathy or biochemical signs of inflammation or infection in the cerebrospinal fluid. Potential control subjects underwent an interview designed to ensure that none of the control group had symptoms of polyneuropathy, including diabetic polyneuropathy. All control participants
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had only mild neurological symptoms and, after diagnostic evaluation, none of them had a neurological disorder or showed signs of polyneuropathy. In all control participants included, the final diagnosis did not imitate polyneuropathy, including diabetic polyneuropathy. Furthermore, the final diagnoses of the control participants were believed to have a small, if any, impact on peripheral nerve function. Control participants were diagnosed with Bell’s palsy (n=9), benign muscle fasciculations (n=2) and idiopathic paresthesia (n=1). All cerebrospinal fluid samples were analysed with biochemical tests and none of the control participants displayed cerebrospinal fluid leukocytosis. Serum HbA1c levels were measured in all control participants to exclude those who
had diabetes. All participants gave informed consent to the study, which was approved by the local Ethics Committee and the Danish Data Protection Agency and performed in accordance with the Helsinki Declaration.
Diagnosis and staging of diabetic polyneuropathy Symptoms of peripheral neuropathy were assessed according to the neuropathy symptom score, which includes sensory, motor and autonomic symptoms of neuropathy. Participants with diabetes were evaluated with a standardized neurological examination performed by a trained neurologist (H.A.) resulting in a neuropathy impairment score, which is a combined score from the evaluation of muscle weakness, tendon reflexes and sensation. A high neuropathy impairment score or neuropathy symptom score reflects more severely impaired nerve function [14,15]. All participants with diabetes underwent nerve conduction studies with transcutaneous stimulation, using standardized recording procedures and electromyography equipment (Keypoint Medtronic, Skovlunde, Denmark). Skin temperatures were held above 32°C. Sensory nerve conduction velocity and compound sensory action potential amplitude were the variables evaluated for sensory nerve
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conduction studies, which were recorded in the dominant arm median nerve and bilateral sural nerves with antidromic stimulation. Motor nerve conduction velocity, distal motor latency, compound motor action potential amplitude and F-wave latency were the variables evaluated for motor nerve conduction studies recorded in the median nerve of the dominant arm and bilaterally in the tibial and peroneal nerves. Participants with median nerve impairment underwent additional sensory and motor nerve conduction studies of the dominant ulnar nerve to exclude carpal tunnel syndrome. Results within the range of mean ± two standard deviations compared with standard reference material were considered normal. Vibration perception thresholds were determined in all participants with diabetes using a computerbased sensory evaluation system (CASE IV, WR, Medical Electronics, Stillwater, MN, USA). Skin temperatures were held between 30 and 35 °C and sensory testing was performed at the dorsal site of the non-dominant first toe and dominant index finger. Diabetic polyneuropathy was diagnosed in participants with diabetes if two or more of the nerve conduction variables or vibration perception thresholds were abnormal: a neuropathy symptom score > 1; a neuropathy impairment score ≥ 2; a vibration perception threshold at the index finger and great toe ≥98th percentile; and abnormal motor or sensory nerve conduction velocity or abnormal compound motor or sensory action potential amplitude in at least two of four nerves: the dominant median nerve (motor and sensory), the non-dominant peroneal nerve or the non-dominant sural nerve [13]. For all participants with diabetes a nerve conduction study rank score was calculated. A high nerve conduction study rank score reflects more severely impaired peripheral nerve function. It was calculated as the sum of individual rankings of z-scores from the following nerve conduction variables: compound action potential amplitude and nerve conduction velocity of dominant median motor and sensory nerves; bilateral peroneal and tibial motor nerves; and bilateral sural sensory
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nerves. A neuropathy rank sum score was calculated for all participants with diabetes in order to rank the participants according to peripheral nerve function. The neuropathy rank sum score for each participant was calculated by adding the individual rank scores for neuropathy symptom score, neuropathy impairment score, nerve conduction study rank score and vibration perception threshold; a high neuropathy rank sum score reflected more impaired nerve function.
Measurement of soluble CD163 in cerebrospinal fluid and serum Blood and cerebrospinal fluid concentrations of soluble CD163 were determined in duplicates by use of an in-house sandwich enzyme-linked immunosorbent assay, essentially as described
previously [16]. In each run, we co-analysed control samples and the serum calibrator with concentrations traceable to purified CD163. Serum was measured on a BEP-2000 enzyme-linked immunosorbent assay-analyzer (Dade Behring, Marburg, Germany) diluted 1:101, and cerebrospinal fluid 1:3 before analyses. The inter-assay imprecision over the latest runs (n=159) was a 5.1% coefficient of variation at concentrations of 1.91 mg/l, and a 7.6% coefficient of variation at 3.83 mg/l. The limit of detection was 6.25 µg/l.
Clinical and standard biochemical measurements Standard biochemical measurements were performed by accredited methods at the Department of Clinical Biochemistry; serum HbA1c concentrations were measured in all participants. Furthermore,
to exclude competing causes to polyneuropathy, the following laboratory tests were performed in participants with diabetes: kidney, thyroid and liver function; immunoglobulin; M-component; and Wassermann reaction test. Cerebrospinal fluid protein concentration was measured in all study participants. Blood lipid concentrations were obtained from patient charts, but this information was missing in two control participants. Information regarding weight, height, gender, age and duration
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of diabetes (when applicable) were obtained for all study participants using patient interviews. In all participants with diabetes and 75% of the control participants, blood pressure was determined. Information on the use of anti-hypertensive, lipid-lowering and antiglycaemic drugs was obtained from patient charts.
Statistical analysis Data were analysed using STATA/IC_12 (StataCorp LP, TX, USA). For non-parametric data a
Kruskal–Wallis test was applied to address independence between groups. For parametric data Student's t-test was used to test for differences between two groups and for non-parametric data the
Mann–Whitney U-test was applied. To evaluate the statistical association between two variables, Spearman’s rank correlation was used. Two-tailed P-values < 0.05 were considered significant and
descriptive data are presented as median (range) values. A 30% difference in cerebrospinal fluid soluble CD163 concentration and a 50% difference in serum soluble CD163 concentration were expected between participants with diabetes and control participants. Based on previous studies of soluble CD163 concentration in cerebrospinal fluid in symptomatic control subjects (M. Stilund, unpublished data) and in serum in healthy control subjects [8], sample size calculations resulted in optimum group sizes of 10 participants to detect the difference in cerebrospinal fluid and 20 participants to detect the difference in serum (0.05 twosided significance level and 90% power).
Results Demographic characteristics of the study population Participant demographics and clinical information are shown in Table 1. The majority of the study population was male (79%). Participants with diabetes and control participants were similar with
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regard to age (P = 0.08), cerebrospinal fluid protein concentration (P = 0.09) and systolic blood
pressure (P = 0.73), while the two groups differed with regard to BMI (P = 0.01), serum HbA1c concentration (P < 0.01), serum total cholesterol concentration (P < 0.01) and number of
participants receiving anti-hypertensive or lipid-lowering treatment (P= 0.02 and P = 0.03,
respectively). Participants with diabetes, with and without polyneuropathy, were similar with regard to gender (P = 0.10), age (P = 0.81), BMI (P = 0.25), serum HbA1c and total cholesterol concentration (P = 0.16 and P = 0.30, respectively), systolic blood pressure (P= 0.06), diabetes duration (P = 0.84) and
number of participants receiving anti-hypertensive and lipid-lowering treatment (P = 0.11 and P =
0.43, respectively). Participants with diabetes and polyneuropathy had a higher cerebrospinal fluid protein concentration than participants with diabetes without diabetic polyneuropathy (P < 0.01). Notably, more participants without neuropathy than with neuropathy received insulin treatment (64 vs 13%; P = 0.02). As expected, the two groups differed in clinical neuropathy scores (neuropathy symptom score: P < 0.01; neuropathy impairment score: P < 0.01).
Comparison of cerebrospinal fluid and blood soluble CD163 levels and their association with peripheral nerve function Cerebrospinal fluid and serum soluble CD163 concentrations were higher in participants with diabetes than in control participants (Table 2). There was a trend towards higher levels of cerebrospinal fluid and serum soluble CD163 in participants with diabetes and polyneuropathy than in participants with diabetes without polyneuropathy [cerebrospinal fluid: median (range) 131 (86– 173) vs 101 (70–190) µg/l, P = 0.08 and serum: 3725 (920–7060) vs 2220 (1130–4780) µg/l, P = 0.06 (Figure 1)].
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When comparing participants with diabetes receiving s.c. administered insulin treatment with participants treated with oral antidiabetic drugs only, there was no difference in the concentration of soluble CD163 in cerebrospinal fluid [median (range): 109 (84–190) vs 105.5 (70–173) µg/l; P = 0.79] or serum [median (range): 2305 (1130–5000) vs 2480 (920–7060) µg/l, P = 0.90,
respectively]. Neurogenic pain was present in four out of eight participants with diabetic polyneuropathy. There was no difference in cerebrospinal fluid or serum soluble CD163 when comparing participants with diabetes and diabetic polyneuropathy with or without pain [cerebrospinal fluid: median (range) 131 (103–145) vs 129.5 (86–173) µg/l, P = 0.77 and serum: median (range) 4555 (1300–7060) vs 2825 (920–4010) µg/l, P = 0.15, respectively].
In analysis of all study participants, cerebrospinal fluid soluble CD163 showed a significant correlation with age, serum HbA1c and total cholesterol concentrations as well as with cerebrospinal
fluid protein levels (Table 3a). Serum soluble CD163 was associated with BMI and serum HbA1c and total cholesterol concentrations. In participants with diabetes cerebrospinal fluid soluble CD163 correlated to cerebrospinal fluid protein and nerve conduction study rank score (Fig. 2) but was unrelated to neuropathy rank sum score or neuropathy symptom score. There was a trend towards a positive correlation between cerebrospinal fluid soluble CD163 and neuropathy impairment score (P = 0.06; Table 3b). There
was no significant difference in cerebrospinal fluid soluble CD163–protein ratio between diabetic participants with and without polyneuropathy [median (range) score 207 (155–308) vs 263 (145– 363) P = 0.09].
Serum soluble CD163 did not correlate to cerebrospinal fluid protein or any measure of peripheral nerve function including nerve conduction study rank score, neuropathy rank sum score, neuropathy impairment score or neuropathy symptom score .
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In participants with diabetes, cerebrospinal fluid protein correlated with neuropathy symptom score (r = 0.51; P = 0.02), neuropathy impairment score (r = 0.67; P < 0.01), nerve conduction study rank score (r = 0.44; P = 0.04) and neuropathy rank sum score (r = 0.63; P < 0.01) but not with age (r = 0.09; P = 0.69).
Discussion To our knowledge, this is the first study of soluble CD163 in both cerebrospinal fluid and serum in people with diabetes and furthermore the first study to assess the relationship between soluble CD163 and neuropathy in diabetes. The findings lend support to the hypothesis that inflammation plays a role in the development of diabetic polyneuropathy. We found a higher concentration of soluble CD163 in cerebrospinal fluid and serum in participants with Type 2 diabetes compared with control participants. Moreover, there was a trend for higher levels of soluble CD163 in cerebrospinal fluid and serum in participants with diabetic polyneuropathy than in participants without it. Cerebrospinal fluid soluble CD163 was related to a marker of peripheral nerve conduction (nerve conduction study rank score: r = 0.42; P = 0.0497) and a trend towards a significant correlation to clinical signs of peripheral nerve impairment (neuropathy impairment score: r = 0.41; P = 0.06) was found.
The increase in cerebrospinal fluid soluble CD163 may be part of an inflammatory response and hence activation of peripheral nerve CD163+ macrophages in people with Type 2 diabetes and polyneuropathy. CD163+ macrophages and microglia are present in the central and peripheral nervous system and are believed to take part in immune-to-brain signalling and anti-inflammatory mechanisms [11]. Elevated levels of inflammatory markers in cerebrospinal fluid, such as tumour necrosis factor-α, may affect the central and peripheral nervous system and hence participate in the
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development of nervous complications in diabetes. Macrophage infiltration and expression of proinflammatory cytokines in the sciatic nerve has been reported in early phases of experimental diabetic polyneuropathy [17] as well as inflammatory cell infiltrations and immunological lesions in sural nerve biopsies in people with Type 1 and 2 diabetes [18]. Systemic inflammation, as seen in Type 2 diabetes, induces cytokine synthesis within the central nervous system [11] and in experimental Type 1 diabetes the presence of brain inflammation is suggested [19]. Soluble CD163 is transported passively across the blood–brain barrier and is also produced intrathecally [20]. The cerebrospinal fluid protein level, a marker of blood–brain barrier permeability, was higher in participants with diabetic polyneuropathy than in those without. Moreover, cerebrospinal fluid soluble CD163 concentration was positively associated with cerebrospinal fluid protein level in participants with diabetes. This may indicate that degeneration of the blood–brain barrier contributes to elevated cerebrospinal fluid soluble CD163 concentration; however, there was no statistically significant difference in cerebrospinal fluid soluble CD163– protein ratio between participants with or without diabetic polyneuropathy. It remains unclear to what extent the increased blood–brain barrier permeability adds to the increased concentration of cerebrospinal fluid soluble CD163 in participants with diabetic polyneuropathy compared with those without. Interestingly, the cerebrospinal fluid protein concentration correlated very closely to all markers of peripheral nerve function (neuropathy impairment score, neuropathy symptom score, nerve conduction study rank score and neuropathy rank sum score), which suggests that blood– brain barrier degeneration, which is accelerated in people with diabetes, plays a role in the development of diabetic polyneuropathy or alternatively reflects the pathogenetic process in diabetic polyneuropathy. Serum soluble CD163 was higher in participants with Type 2 diabetes than in control participants (Table 2) which is in accordance with former findings showing increased serum cytokine levels and
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hence subclinical inflammation in people with Type 2 diabetes [2,21–23]. Chronic hyperglycaemia activates macrophages [12] and may lead to elevated soluble CD163 levels. Adipose tissue is an important source of inflammatory factors in people with Type 2 diabetes [24] and a relationship between serum soluble CD163 and obesity in people with and without Type 2 diabetes has been shown [22]. In the present study, serum soluble CD163 was significantly associated with BMI (r = 0.4051; P = 0.0175). Nevertheless, the group of participants without diabetic polyneuropathy had the highest BMI, so disparity in BMI cannot explain the association between soluble CD163 and impaired nervous function.
Cerebrospinal fluid soluble CD163 levels correlated with the nerve conduction study rank score, suggesting an association between impaired peripheral nerve conduction and the inflammatory response in the peripheral and central nervous system. Inflammation may play a role in the development of diabetic polyneuropathy and soluble CD163 may be a biomarker reflecting the severity of diabetic polyneuropathy. Inflammatory markers correlate with non-inflammatory polyneuropathy severity [25] and inhibition of tumour necrosis factor-α, a pro-inflammatory mediator, has been shown to improve nerve conduction velocities and density of myelin nerve fibres in experimental diabetic polyneuropathy [26]. Hyperglycaemia-induced metabolic changes lead to dysregulation of cytokine control and this may lead to degeneration and reduced regenerative abilities in peripheral nerves [27,28]. Hyperglycaemia is believed to induce neurotoxicity via a series of pathways [29]; hence the hyperglycaemia itself may explain some degree of nerve degeneration seen in the present study. There is evidence to suggest a link between inflammation and painful diabetic polyneuropathy [3,30]. The present study offers no support to this notion; however, the number of participants
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included is too small to allow any conclusion about the impact of soluble CD163 on the development of pain. The main limitation of the present study is its small size and low number of study participants, which warrants larger studies in future; however, as a hypothesis-generating study, the findings add to the knowledge of the role of inflammation in diabetic polyneuropathy and, in future studies, the mechanisms should be studied in more detail. The control participants were chosen from a group of patients with mild neurological symptoms and this may have affected the results. Because of the limited number of participants we were unable to evaluate participants with asymptomatic diabetic polyneuropathy, but it would have been very interesting to evaluate if this had any effect on the soluble CD163 concentrations in cerebrospinal fluid or serum. Furthermore, because we focused on large fibre function only, the present study allows no conclusion as to whether soluble CD163 has any role in small fibre neuropathy. It is noteworthy that cerebrospinal fluid soluble CD163 only correlates with one marker of peripheral nerve impairment (nerve conduction study rank score); however, nerve conduction abnormalities may more closely reflect the severity of polyneuropathy than clinical examinations (neuropathy impairment score) or presence of neuropathy symptoms (neuropathy symptom score ).
In conclusion, in the present study we found higher levels of soluble CD163 in cerebrospinal fluid and serum in participants with Type 2 diabetes than in control participants. There was a trend for a higher level in neuropathic participants and cerebrospinal fluid soluble CD163 was associated with impaired peripheral nerve conduction. This is, to our knowledge, the first report of an association between soluble CD163 and a late diabetic complication. Future studies are needed to explore whether the innate immune system is activated in the central or peripheral nervous system in people with diabetes and, if so, how this may be related to peripheral nerve function.
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Funding sources The Department of Clinical Medicine, Aarhus University, Denmark, the BEVICA Foundation and the Danish council for strategic research (TRAIN10-092797) are acknowledged for funding of salary and biochemical analysis.
Competing interests H.J.M. has received royalties from IQ-products, The Netherlands. All other authors have no competing interests to declare.
Acknowledgements We thank our laboratory technician, Kirsten Bank Petersen, for excellent technical assistance.
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FIGURE 1 The concentration of sCD163 in patients with Type 2 diabetes with and without neuropathy and
in control participants in (a) cerebrospinal fluid and b) serum. The three groups differed significantly with regard to soluble CD163 in cerebrospinal fluid (P
