sCD163 in ALS Patients
Condition: Amyotrophic Lateral Sclerosis
MeSH terms: Motor Neuron Disease; Amyotrophic Lateral Sclerosis; Sclerosis
Study type: Observational

Summary: 
      Amyotrophic lateral sclerosis (ALS) is a fatal disease with progressive muscle weakness
      leading to severe disability and eventually death.Since the diagnosis relies on clinical
      features and electromyographic abnormalities, which may occur rather late in the disease
      course, there is a need to identify diagnostic tests that can confirm or exclude the
      diagnosis of ALS in the earlier phase of the disease.

      More recently, there are studies suggesting neuroinflammation to play a role for the
      development of ALS. Cluster of differentiation 163 is found to be up regulated in a large
      range of inflammatory diseases.

      At the investigators lab, pilot data (Kallestrup M et al, unpublished data) has shown
      promising results. There was an increased level of cluster of differentiation 163 (sCD163) in
      cerebrospinal fluid in 7 patients with ALS compared with controls.

      The purpose of the investigators study is to define the concentration of sCD163 in the
      cerebrospinal fluid and serum in patients with ALS compared with controls (patients with
      unspecified neurological symptoms). Furthermore, the investigators will define the
      concentrations of protein, glucose, immunoglobulin G index and other factors in the spinal
      fluid.
    

Detailed description: 
      Introduction Amyotrophic lateral sclerosis (ALS) is a fatal disease with progressive muscle
      weakness leading to severe disability and eventually death. It is the most common form of
      motor neuron diseases and involves both the upper and lower motor neurons. The median
      survival from debut of symptoms to dead is approximately 3 years.

      Since the diagnosis relies on clinical features and electromyographic abnormalities, which
      may occur rather late in the disease course, there is a need to identify diagnostic tests
      that can confirm or exclude the diagnosis of ALS in the earlier phase of the disease.

      ALS is a neurodegenerative disease, however the pathogenesis is not well established. The
      causes are probably multifactorial and a complex interaction between genetic factors and
      dysfunction of vital molecular pathways concerning the dominantly inherited c9orf72 gene and
      impaired glutamate uptake in astrocytes (EAAT2-receptor) from the synaptic cleft and thereby
      glutamate excitotoxicity. This glutamate excitotoxicity may cause oxidative stress,
      voltage-gated persistent sodium channels and activation of microglia resulting in secretion
      of proinflammatory cytokines.

      More recently, there are studies suggesting neuroinflammation to play a role for the
      development of ALS. A study from 2012 (a mouse model of ALS, that overexpress mutant
      superoxide dismutase (mSOD1)), has documented that microglia can both protect and injure the
      motor neurones depending on which phenotype of the microglia is activated. It seems like it
      is a continuum between the protective M2 phenotypic state (disease onset) and the neurotoxic
      M1 phenotypic state (disease end-stage). Furthermore, at disease onset the mice expressed
      higher levels of Ym1, CD163 and brain-derived neurotrophic factor (markers of M2).

      Of other molecular mechanisms, it is worth to mention the structural and functional
      abnormalities of mitochondria, impairment of axonal transport systems and endosomal
      trafficking and induction of the endoplasmic reticulum stress response although they appear
      to be secondary events in ALS. The net result is degeneration of motor neurones in cerebral
      cortex, the cranial nerve nuclei, the anterior horn of the spinal cord and the corticospinal
      tracts. The only present treatment available is Riluzole that has proven to extend life or
      time to onset of serious respiratory problems in patients with ALS. The mode of action for
      Riluzole still has to be determined, possibly the dynamic is an inhibition of glutamate
      release from the presynaptic neuron. (1,2,3,4,5,6) Though it seems that the inflammatory
      process is a secondary event, it seems to be critical for neuronal degeneration. In a study,
      ALS pathology was sought to be reduced through central nervous system targeted glucocorticoid
      and succeeded by reducing brainstem pathology in mouse models of ALS. Of different
      biomarkers, inflammatory markers have also been tested: the interleukin-1 family cytokines.
      Among them only total interleukin-18, its endogenous inhibitor interleukin-18BP, and the
      active form of the cytokine (free interleukin-18) were significantly higher in the ALS
      patients than in controls. The cytokine showed no correlation to the different clinical forms
      of ALS or the clinical setting of the disease. Cytokines have also been tried as
      neuroprotective agents. Erythropoietin (EPO) is known as the growth factor that maintains the
      number of circulating erythrocytes. However, the biological role of EPO has been expanded to
      other cells, such as neurons, microglia, and astrocytes in the brain. EPO seems to suppress
      the proinﬂammatory cytokines during disease progression, maintain the anti-inﬂammatory
      cytokines until the late symptomatic stage and finally seems to delay symptom onset and
      preserve number of motor neurons. (7,8,9,10) Cluster of differentiation 163 (CD-163) - a new
      way to monitor inflammation in ALS? CD-163 is a scavenger receptor (recognizes and uptakes
      macromolecules) which marks the monocyte-macrophage activation. CD163 works for macrophages
      as a receptor for hemoglobin-haptoglobin complexes. The soluble form of the receptor is
      called sCD163 and is found in plasma and cerebrospinal fluid, where it is found to be up
      regulated in a large range of inflammatory diseases.

      Strongly increased values (>20mg/l) is seen in diseases like macrophage activating
      syndrome,hemophagocytic syndrome and acute hepatic failure. Furthermore, the level of sCD163
      reflects the activity of the disease. Patients with increasing and high values are found to
      have unfavorable prognosis. Increased values (5-20 mg/l) are seen in hepatic disease, sepsis
      and Gaucher's disease. Septic patients with values greater than 10 mg/l have a 10 times
      greater risk of fatal outcome. Values which is increased a little (3-5 mg/l) is often seen in
      inflammatory of infectious conditions and in cancer. (11,12,13,14) Pilot data At the
      investigators lab, pilot data (Kallestrup M et al, unpublished data (15)) has shown promising
      results. There was an increased level of sCD163 in cerebrospinal fluid in 7 patients with ALS
      compared with controls. Furthermore, both definite and probable ALS patients have an
      increased level of sCD163. These results suggest a larger study to be performed evaluating
      patients with ALS at early as well as later stages of the disease.

      Hypothesis Patients with ALS have up-regulated levels of sCD-163 in their cerebrospinal fluid
      and serum compared with healthy subjects and the level is related to the severity of the
      disease.

      Purpose To define the concentration of sCD-163 in the cerebrospinal fluid and serum in
      patients with ALS compared with controls (patients with unspecified neurological symptoms).
      Furthermore, we will define the concentrations of protein, glucose, immunoglobulin G index
      and other factors in the spinal fluid.

      Method Study population The study population will be all patients with established and
      suspected ALS at the Department of Neurology, Aarhus University Hospital.

      Patients with established ALS who participate will undergo lumbar puncture as a part of this
      study. Subjects suspected to suffer from ALS will undergo lumbar puncture as a part of their
      diagnostic work up and the level of sCD163 in their cerebrospinal fluid will be analysed.

      At the Department of Neurology, Aarhus University Hospital there are approximately 80
      patients with established ALS. We expect that 50 of these patients are willing to participate
      and are eligible for inclusion (31-08-2015). Furthermore, we expect to include 8 patients who
      will be diagnosed with ALS at Department of Neurology, Aarhus University Hospital during the
      study period.

      The controls include patients with various neurological symptoms or diseases who will undergo
      a lumbar puncture at the Department of Neurology at Aarhus University Hospital as part of
      their diagnostic work-up irrespective of this study. In the final analyses only controls will
      be included who end up having either no neurological disease or a neurological disease which
      is believed not to involve the spinal fluid. We expect to include 40-50 controls.

      Recruitment Participants will receive written and oral information about the study. The oral
      information will take place at Department of Neurology, Aarhus University Hospital. Moreover,
      the pamphlet "Forsøgspersoners rettigheder i et sundhedsvidenskabeligt forskningsprojekt"
      will be handed out. Consultants at the department of neurology will give the information, to
      patients with suspected ALS. Controls and patients with established ALS will receive
      information from the doctors at the department of Neurology and research year student Anne
      Sofie Vinther.

      It is possible to bring a companion and the participants are offered at least 24 hours to
      consider participation.

      Access to patient records will be requested, which will be provided in the written
      information material.

      If new knowledge about disadvantages of participating in the study appears the participants
      will be informed immediately. Moreover, the participant will be informed if evaluations
      during the study bring knowledge about the general health status of the patient, unless this
      has been clearly rejected by the patient.

      By the end of the study it is possible for participants to contact the study coordinator to
      get information about study results.

      All participants will receive information about the research project and will be asked to
      give their content to participation. The participation implies extraction of 5-6 ml spinal
      fluid and a blood sample (30ml), which will be used for analysis of sCD-163.

      Participants will not receive any form of payment, but can receive compensation for
      transportation.

      Inclusion criteria:

      ALS Patients diagnosed with ALS and patients suspected to have ALS. Age >18 Controls Age >18

      Exclusion criteria (for all groups):

      Acute infections including neuroinfection Other disorders known to have elevated levels of
      sCD163 Disorders or treatments that contraindicate a lumbar puncture. Other established
      neurological disorders. At all times during the diagnostic work-up and consequently in the
      research project we will do this with respect for the patients mental and physical condition.
      The research project will be carried out according to the Act on Processing of Personal Data
      and, furthermore, the project will be submitted to the common regional information system for
      Region Midtjylland.

      Analysis The spinal fluid and the serum will be analysed at the Department of Clinical
      Biochemistry, Aarhus University Hospital. The spinal fluid and blood samples will be
      deposited in a research biobank at Aarhus University Hospital. It is planned that all
      biochemical analyses will be performed when all samples are collected. At the end of the
      project, the samples will be pseudo anonymised. Due to the possibility of developing new
      knowledge in this area, the samples will be deposited in the biobank 5 years after this
      project has been finished.

      The concentrations of sCD-163 in spinal fluid and serum will be analyzed by already
      established techniques at the Department of Clinical Biochemistry, Aarhus University
      Hospital.

      Examination The examination consists of a medical history and a clinical examination. We use
      the Revised ALS functional rating scale (ALSFRS-R), which is at rating instrument for
      monitoring the progression of disability in patients with ALS. Information from the medical
      record will be included and used in this project including past medical history, actual use
      of drugs, results of biochemical analyses and brain imaging, results from the ALSFRS-R,
      results from the electromyography, abnormalities at the clinical examinations and the
      conclusions from the diagnostic work-up.

      Power and Statistical analyses Since the investigators pilot data (n=7) showed a 50% increase
      in the level of CD163 in the cerebrospinal fluid in ALS patients inclusion of 40 patients is
      expected to enable detection of smaller difference also in serum and relations between CD163
      and clinical findings.

      The program ANOVA will be used for statistical analyses. Parametric or non-parametric tests
      will be used depending of the normal distribution of data. In all statistical analysis a 5%
      level of significance will be used.

      Perspectives It is of importance to identify prognostic biomarkers in ALS. The pathology of
      ALS is still unknown but it seems that inflammation is a crucial factor in the process. In
      several studies, sCD163 has shown to be up regulated in a large range of inflammatory
      diseases and together with the investigators promising pilot data sCD163 is a potential
      marker for ALS. (4) The assessment of drug efficacy in ALS remains clinically based, relying
      on measuring the rate of disease progression through the utilization of the ALS rating
      scale-revised (ALSFRS-R). Such clinical scales may be insensitive, especially in the early
      stages of the disease process. The development of reliable quantifiable biomarkers remains
      elusive in ALS, and development of prognostic biomarkers would be crucial for effective
      evaluation of a therapeutic agent in the early stages of development.

      Ethical consideration The tests performed on the patients involved in this research project
      are lumbar puncture and blood samples.

      It involves some risks to perform a lumbar puncture, which include post lumbar puncture
      headache with an incidence of 12 %, most common in younger patients. Most frequently, the
      headache has a short duration and it may be cured with application of a blood-patch.
      Moreover, lumbar puncture can cause temporary lumbar pain and an accumulation of blood
      underneath the skin. There is a very low risk of bleeding in the epidural space with
      compression of spinal nerves and an even lower risk of infections (meningitis or
      meningoencephalitis). However, it is important to underscore that lumbar puncture is part of
      routine diagnostic workup at the Department of Neurology at Aarhus University Hospital.

      In consideration of the low risk involved in this project, the bad prognosis for the disease
      and the potential to improve diagnosis and treatment for future patients with ALS we consider
      this project to be ethically acceptable.

      Publication Positive, negative as well as inconclusive results from this study will be
      published. The results will be written in an article for publication in an English written
      peer reviewed journal.
    

Intervention name(s): lumbar puncture for spinal fluid and blood sample
Intervention type(s): Procedure
Intervention description(s): lumbar puncture for spinal fluid and blood sample
Sampling method: Non-Probability Sample
Study population: 
        Patients with ALS, treated or newly diagnosed
      
Biospecimen description: 
      spinal fluid and blood sample for identification of different factors
    
Keywords: Amyotrophic Lateral Sclerosis; spinal fluid; CD163