Amyotrophic Lateral Sclerosis (ALS) is an aggressive, deadly disease. ALS leads to destruction of the neural pathways which control the conscious movements of the muscles. This destruction leads to muscular dystrophy with increasing difficulties in moving, breathing, swallowing, and speaking. In the last phase of an ALS patient's life it is necessary with respiratory therapy in order to breathe. In average an ALS patient lives 3 years from the time he or she gets the diagnose.

The cause of the disease is still unknown and there is currently no treatment which can stop the progression of the disease. Former clinical studies have indicated that the innate immune system and in particular the complement system plays a significant role in the progression of ALS. The complement system, which is activated in cascades, is part of the innate system but participates in the innate as well as the acquired immune system. Former clinical trials have been characterized by limited knowledge about both the complement system as well as to how it is measured.

Today it is possible to measure directly on the different components of the complement system and to understand its contribution to the overall immune response. It is also possible today to detect defects of the complement system. All these progressions are the foundation for this project which is carried out in close cooperation with one of the world's leading researchers in the complement system, professor Peter Garred from Rigshospitalet.

The aim is to make a national research project about ALS in order to investigate the role of the innate immune system, and especially the complement system, in patients with ALS.

In the long term the hope is, that this will lead the way to a targeted and effective medical treatment to the people affected by this grave disease.

Amyotrophic lateral sclerosis (ALS) is a progressive, deadly, neurodegenerative disease which affects the upper and lower motor neurons. This leads to profound muscular dystrophy, hyperreflexia, fasciculations and paresis of the bulbar as well as the skeletal musculature. ALS causes increasing physical fatigue and the patients soon become bedridden and respiratory insufficient.The diagnosis ALS is made according to the El Escorial revisited. Often clinical and neurophysiological tests must be repeated (1-4).

In Denmark the incidence of ALS is 1-2/100.000 and the prevalence is 4-6/100.000. The average survival time from the time of the diagnosis is 3 years but with great variance. (5+6)

Today the pathogenesis is still unknown and no treatment can stop the progression of ALS. Treatment with riluzole seems to prolong the median time of survival for 2 or 3 months (7).

Most likely, a future medical treatment requires a better understanding of the pathogenesis as well as the pathophysiology of ALS. This present study aims to do so based on the hypothesis that ALS partially or fully is caused by complement activation.

The complement system is a complex system consisting of proteins in plasma as well as membrane bound proteins which together complement the antibody-based immune system. The complement system is a self-perpetuating cascade system which is activated through different pathways. It works by opsonisation where complement proteins bind to microorganisms to activate and target granulocytes, monocytes and macrophages. The complement system also causes cytolysis of microorganisms via MAC (membrane attack complex) by activation of the mast cells. It also inactivates and eliminates burned out immune complexes as well as performing apoptotic renovation.

A recent pilot study of Neuromyelitis Optica has shown how the complement system play a central part in the pathogenesis of a disease that previously was wrongfully perceived as an early stage of Disseminated Sclerosis. These findings resulted in medical treatment with anticomplement (equlizumab) with promising results (8). It is possible that similar pathogenetic mechanisms could be the molecular basis of ALS.

Different research groups have tried to illuminate how the immune system is involved in the progression of ALS (9-35). Several studies support the hypothesis that the complement system is of crucial importance for the onset and progression of ALS. (9-28)

In several clinical trials with ALS plasma it is concluded that ALS plasma is cytotoxic when incubated with healthy red blood cells or healthy nerve tissue. Some research indicates that the cytotoxicity is caused by the complement system. The results cannot, however, be reproduced consequently in all clinical trials. These trials were conducted decades ago where the methods of detecting complement activity were limited. (9-14)

Several animal trials indicate that ALS starts in the neuromuscular junctions (NMJ) and therefore should be considered a distal axonopathy rather than a central neurological disease, which today is the general perception of ALS. (27-31)

The purpose of the project The aim is to increase the knowledge of the pathophysiology of the disease ALS as this possibly may lead us closer to a targeted medical treatment.

The project group wants to investigate if a previously found, unique cytotoxicity in the ALS plasma can be retrieved (9-14). If this is the case, then the modern methods of today make it possible to detect whether the immune system in general, and in particular the complement system, is causing this cytotoxicity.

The establishment of a national research project about ALS and the complement system by making a research biobank with systematically collected blood and spinal liquid from ALS patients from all over the country will ensure the opportunity to find out if the complement system plays a role in the onset and progression of ALS.

Furthermore a pilot study will be conducted with the purpose to investigate if there is complement activity in the NMJ in patients with ALS as it has been shown in a study of ALS mice. (28)

The hypotheses

1. ALS is characterized by an abnormal immune response with cytotoxicity and increased complement activity in plasma which is revealed by the following:

   1. Plasma from patients with ALS shows increased haemolytic activity when incubated with red blood cells from healthy subjects.
   2. The complement activity is increased in the plasma from ALS patients compared with plasma from neurologically healthy subjects and subjects with other neurological disease.
   3. The haemolytic activity in the plasma from ALS patients is correlated with the complement activity.

2. Spinal liquid from ALS patients contains increased complement activity compared with spinal liquid from neurologically healthy subjects and from subjects with other neurological disease.

Patients, materials and methods:

Clinical trial 1(CT1): Haemolytic activity and the complement system in ALS plasma

Number of subjects: 25 patients with ALS, 25 patients with other neurological disease, 25 healthy volunteers

The course of CT1: A blood sample is taken from each patient. The red blood cells and the liquid part of the blood, the plasma, are separated. The red blood cells from different subjects are incubated in an other subjects plasma.The same trial course is repeated after inactivation of the present complement system both by heat and by anti-complement.

Clinical trial 2(CT2): Case-control study aiming to mapping the complement system

Number of subjects:100 patients with ALS, 100 patients with other neurological disease and 100 neurologically healthy patients

The course of CT2:Blood samples and cerebrospinal fluid are prepared and then freezed in a research biobank. Then the samples from the 3 groups of subjects are analysed and compared focusing on the complement system: The complement activation potential is measured in the biological material. A cytokine profile is made as well as mapping the acute phase reactants by multiplex assays. Furthermore the RNA expression profile is made on a cell pellet stabilised with RNA later.

Clinical trial 3(CT3): The complement system of ALS patients over time - a cohort study Number of subjects:20 patients with ALS (subset from CT2)

The course of CT3: Every sixth month the course from the CT2 is repeated. The activity of the complement system in each patient with ALS is analyzed as the disease progresses.

Clinical trial 4(CT4): Searching for complement activity in the NMJ of ALS patients

Number of subjects:10 patients with ALS

The course of CT4: The muscle biopsies are taken and immediately brought to the Dep. of Pathology at Rigshospitalet. Then thin layers of tissue are stained in order to analyze the muscle fibers and the NMJ as well as detecting presence of complement activity.

Conducting the study The project consists of four clinical trials. Inclusion of the subjects is done together with staff in ALS outpatient clinics at hospitals all over the country and subjects for the control groups are included according to the list of inclusion sites below.

Inclusion sites:

ALS outpatient clinic, Neurological clinic, Rigshospitalet Glostrup (CT1+2+3+4) Contact: Chief physician Elisabeth Elmo Neurological Clinic, Rigshospitalet Glostrup (neurological control group, CT1+2) Contact: Professor, Chief physician, dr.med. Rigmor Højland Jensen Neurosurgical Clinic, Rigshospitalet (neurological control group, CT2) Contact: Professor, chief physician, dr.med. Marianne Juhler ALS outpatient clinic, Neurological Dep., Bispebjerg Hospital (CT2) Contact: Chief physician Merete Karlsborg ALS outpatient clinic, Neurological Dep., Roskilde Hospital (CT2 + 3) Contact: Chief physician Helle Thagesen ALS outpatient clinic, Neurological Dep., Odense University Hospital (CT2) Contact: Chief physician, dr.med. Matthias Bode ALS outpatient clinic, Neurological Dep., Aarhus Hospital, Nørrebrogade (Clinical Trial 2) Contact: Chief Physician, ph.d. Anette Torvin Gildhøj Private Hospital, Brøndby (Neurologically healthy control group, Clinical Trial 2) Contact: Anaesthesiologist Niels Anker Pedersen

Power calculations CT1: Haemolytic activity and the complement system in ALS plasma The study Overgaard et al. (18) found a mean difference of about 0,20 (SE 0,052 in the ALS group, N=20, SD 0,22) in the absorbance (415 nm and 5 hours of incubation) between ALS patients and healthy bioanalysts. With α=0,05 og beta=0,20 corresponding to power 0,80 we need to include 21 subjects. As possible drop outs and technically failed are considered tests the investigators choose to include 25 subjects in each group. (36)

CT2: Case-control study mapping the complement system The number of subjects in each group is in this case-control study calculated with α=0,05 The investigators compare the complement activation potential of 3 groups with same amount of subjects in each. In healthy subjects the complement activation potential is 100 % with a normal area ranging from 50-150 % and where the prevalence of low complement activation potential (under 50 %) is under 10 %. With power =0,80 it is calculated to be necessary to include 100 subjects in each group. Hereby it is possible to find statistically significant differences between the groups corresponding to an odds ratio of 2,3, which would correspond to 20 % of ALS patients having a low complement activation potential caused by increased complement activity. (36)

CT3: The complement system of ALS patients over time - a cohort study This is a hypothesis generating study. It is expected that the included 20 ALS patients in this cohort will be a subset from Clinical Trial 2. As a control group at baseline the neurologically healthy control group from Clinical Trial 2 will be used.

CT4: Searching for complement activity in the NMJ of ALS patients There is no previous studies describing the complement activity in the NMJ in living humans. It is therefore not relevant to make a calculation of power.

Data processing CT1: Haemolytic activity and the complement system in ALS plasma Comparing the degree of haemolysis between the ALS patients and the control groups t-test and one way ANOVA are used. For calculating the cut-off values the investigators use receiver operating characteristic (ROC) curves.

CT2: Case-control study mapping the complement system Comparing the concentration of complement and the complement activation potential between ALS patients and the control groups t-test and one way ANOVA are used. In order to calculate the odds for low complement activation potential in the ALS group compared with the control groups the investigators use logistic regression. For calculating the cut-off values receiver operating characteristic (ROC) curves are used.

CT3: The complement system of ALS patients over time - a cohort study As in Clinical Trial 2. Furthermore, regression analysis of the complement activity as a function of time since the onset of ALS, gender, age, subtype of illness and disease progression are conducted.

CT4: Searching for complement activity in the NMJ of ALS patients As in CT2. The degree of complement deposition and muscle pathology is described qualitatively and a blinded scoring in "normal", "light degree" and "severe degree" of changes is conducted. This will be compared quantitatively with 2 x K tables and non-parametric statistics.

Dissemination of results The results of the project will be published in international peer reviewed, journals. Both positive and negative findings will be published.

Perspectivation With the establishment of a big national ALS research biobank it will be possible to conduct many future research projects. Continuous research in ALS is paramount for ALS patients nationally as well as internationally in order to maintain hope for an efficient medical treatment for this aggressive disease is found in the future.

Inclusion Criteria:

• For ALS group:Diagnosed with the diagnose category "certain ALS" or "likely ALS according to the El Escorial rev. diagnose criteria
• For Neurological control group: Referred to neurological department to be examined for acute or chronic headache or referred to get a lumbar perfusion test performed.

Exclusion Criteria:

• For all groups (Clinical study 2-3): permanent contraindication for having a lumbar puncture performed
• For Neurological control group: Known with chronic inflammatory disease or autoimmune disease.
• For healthy control group (clinical study 1): Known with any disease
• For healthy control group (clinical study 1): Taking daily medication
• For Neurologically healthy control group (Clinical study 2): Known with neurological disease
• For Neurologically healthy control group (Clinical study 2): Known with chronic inflammatory disease or autoimmune disease.

ALS group: Patients diagnosed with certain or likely ALS

Neurological control group:

Patients being examined for cronical headache or being referred to hospital to get a lumbar perfusion test performed.

Neurologically healthy control group:

Clinical study 1: Healty employees from Rigshospitalet Clinical study 2: Neurologically healthy patients having planned orthopaedic surgery performed in spinal anaestesia.