Norsk Forskning/Norwegian Research studies: Forskningsprotokoll for studien «Autoantistoffer ved kronisk utmattelsessyndrom (ME)» av Tor Lea (NMBU)

[with English description and protocol] Dette innlegget gir beskrivelse av forskningsprotokollen (lang; eng) for en antistoffprofilering av pasienter med Myalgisk Encefalopati (ME), også kjent som kronisk utmattelsessyndrom ME/CFS. Prosjektleder er professor Tor Lea, Institutt for kjemi ved Norges miljø og biovitenskapelige Universitet (NMBU) i Ås, Akershus. Studien har tittel «Autoantistoffer ved kronisk utmattelsessyndrom (ME)», med vitenskapelig tittel; «Autoantistoffprofilering av ME/CFS-pasienter».

Forskningsstudien var planlagt å starte 22 februar 2015 og forventet avsluttet 25 juni 2015. Studiens hovedmål/hensikt er å vurdere om der kan finnes en biomarkør ved å undersøke en autoantistoffprofileringstest, bestående av 36 ulike autoantistoffer. Studien ble godkjent av REK 19 mars 2015.

Beskrivelse av prosjektsøknaden innsendt REK kan du finne her.

English introduction:

This blogpost will provide you with the research protocol for the study: «Autoantibody profiling in myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS)«. The study started 22 Feb 2015 and ends 25 Jun 2015. The principal investigator of this study;  Prof. Tor Lea at the Norwegian University of Life Sciences (NMBU), Ås, Norway.

Kortlenke: http://wp.me/p1wvHk-1EF

Lra_presentasjon_april2016_3

Forskningsprotokollen er som følger:

Project outline

Autoantibody profiling in myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS)

Relevance

Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS) is a debilitating condition for which there is currently no established etiology and no standard treatment. The Centers for Disease Control and Prevention estimates that the prevalence of CFS in the Western population varies from 0.2 –2.5%, depending on the diagnostic criteria used.

Various criteria exist that allow ME/CFS to be diagnosed based on a patient’s subjective symptoms.

However, no single biomarker has been detected that can be reliably used for diagnosis. This significantly impedes the ability of physicians to distinguish patients with ME/CFS from people who may suffer from diseases with similar symptoms. The lack of reliable biomarkers in ME/CFS also makes disease progression much more difficult to track, both in the clinic and among researchers studying disease mechanisms. Consequently, there is an urgent need for the development of tests that may better allow both diagnosing and evaluation of patients with ME/CFS.

Autoantibody profiling may help distinguish patients with ME/CFS from other diseases

Because ME/CFS shares characteristics with many diseases considered autoimmune or immunemediated (e.g., symptom improvement after immunosuppressive therapy, followed by relapse), it has been increasingly studied with the same or similar tools – and in a similar context – as diseases considered to be autoimmune (1).

A number of studies have reported higher than normal levels of various autoantibodies in ME/CFS.

Yet levels of these individual autoantibodies in ME/CFS differ widely across studies depending on the methodology used and the cohort at hand. Consequently, single autoantibody testing in ME/CFS appears to be of limited use so far.

Among others, Poletaev et al. have published observations that pattern-based autoantibody tests may provide a clearer picture of disease in patients that do not test positive for single autoantibody deviations (2). These pattern-based assessments simultaneously include a battery of different autoantibodies in a given individual. Dysfunction associated with a particular autoantibody is not calculated based on an absolute range, but is instead based on the autoantibody level as compared to other tested autoantibodies in the same individual. The latter may be of importance because there are several findings of different subsets of the immune system in ME/CFS being up or down regulated, but with few clear and consistent results across studies (3,4).

To our knowledge, these pattern based autoantibody tests have not yet been used in ME/CFS. It is possible that these broad-spectrum tests may allow for better assessment of the systemic dysfunction characteristic of ME/CFS.

Further, for decades, pattern-based – or individual – autoantibody testing has served as an early and sensitive detector of disease. One study by Poletaev et al., which used the same methodology as in the current study, detected thyroid autoantibodies many years before the development of clinically apparent disease. Thus, in this instance, Poleatev et al’s test served as a good predictor of who would later develop thyroid disease (5).

Recently, a similar dynamic was reported in patients with Sjögren syndrome. The majority of the patients in the study who eventually developed Sjögren syndrome had detectable levels of the autoantibodies in question as long as 18 years before symptom onset – with levels increasing over the years until that time (6).

It is thus of interest to explore whether these early and sensitive markers of autoimmune disease may also serve as reliable markers of dysfunction in ME/CFS. This could aid in the diagnosis and evaluation of the disease process.

Autoantibody profiling – description of the test to be used in this study

The medical research centre Immunculus, based in Moscow, Russia, offers autoantibody-profiling tests, two of which will be used in the current study. The autoantibody profiling tests were developed in the former USSR after the Chernobyl disaster, in order to help investigators screen individuals at radioactively polluted areas. The tests were intended to help reveal various possible forms of associated pathologies at early stages.

Over the years Immunculus has collaborated with different institutions in Russia and internationally, such as the Russian State Medical University, the Moscow Regional Scientific Research Institute of Obstetrics and Gynecology, the Peoples’ Friendship University of Russia, the Research Institute of Pediatrics and Pediatric Surgery, Bakulev’s Institute of Cardiovascular Surgery and Kazan State Medical Academy. All institutions have implemented in clinical laboratory practice the immunologic diagnostics techniques developed by Immunculus under the general title «ELI-Tests» (short for Enzyme Linked Immuno Test). Furthermore, Immunculus cooperates with scientists from Israel, Mexico, the USA, the UK, Italy, Greece, Germany, India, Ukraine and Azerbaijan. Alexander Poletaev, a leading researcher at this centre, has published several papers on these tests and related issues. (See: http://immunculus.ru/en/node/4 )

The symptoms characteristic of ME/CFS appear to involve multiple physiological systems. The ELItests used in this study offer the additional benefit of connecting the production of certain autoantibodies to dysfunction in particular body systems or organs. While these connections are not definitive, they may still provide clues about the ME/CFS disease process. For example, in other studies testing for some of the same autoantibodies as those in the Immunculus’ kits, antigens to dsDNA have been associated with components of the cell nucleus – a relationship that could indicate virus-induced activation of apoptosis (7,8). Similarly, excess of Abs against β2-Glycoprotein I is a marker of anti-phospholipid syndrome which may be the cause of thrombosis in different organs and leads to miscarriages, strokes, and infarctions9. Similar relationships between autoantibody production and body systems are postulated to exist between the other 34 autoantibodies that form the basis of the ELI-kits that would be used in this study. (See attached: ”Price list eli test kit” for description of the different tests)

Each individual autoantibody tested for with the kits is assigned to one of three ranges associated with different levels of disease/health. The ranges are empirically based. Autoantibodies that fall into a green range are associated with normal levels of immune reactivity. Those that fall into a yellow range can be interpreted as demonstrating a weak deviation from normal reactivity. Autoantibodies levels that fall into a red range are correlated with prominent deviations from normal reactivity.

These ranges should allow us to better assess not just which autoantibodies may be present in a particular subject, but also the extent to which they may be generating dysfunction.

It should be noted that the predictive and diagnostic value of several of the different autoantibodies included in the ELI-tests have not been published in the Medline indexed literature. However, more than 7000 individuals have been subjected to these tests in the Russian institutions mentioned above. Several of these results have been reported in twelve PhD dissertations in the Russian language. (See attached: Clinical_Studies_ELI-P-Complex_Summary_ENG.pdf)

We see the use of these tests in the current study as a first step in assessing whether these multiparametric autoantibody tests may demonstrate dysregulation in ME/CFS. If prominent aberrations in various autoantibody levels are seen in ME/CFS, a more comprehensive exploration of the validity and potential of these tests will subsequently be undertaken.

Objectives

Primary objective

Measure the number of different autoantibodies in ME/CFS patients that are above or below the adjusted normal range. Normal ranges will be adjusted to the individual mean total autoantibody level (see below). Further assess the extent to which these deviations differ from that of control subjects and/or patients who are fatigued but do not fulfill ME/CFS criteria, and assess if the deviations can be a marker of disease severity.

Secondary objectives

Assess whether the total level of autoantibodies (i.e. the 36 autoantibodies to be tested for in this study) can serve as an indicator of disease severity. (i.e. ascertain if there is a linear, exponential or biphasic correlation between the autoantibody level and disease state).

Assess whether deviations in specific autoantibodies are associated with specific structures or organs in ME/CFS patients.

Methods

Subjects

This study will use blood already obtained from subjects in the IRB-approved Biobank: ME/CFS – tematisk biobank. Details on the biobank will be added…

Study material

Serum blood without coagulants will be used in this study.

Analysis

The autoantibody profiling tests will be done in the Immunuculus Research centre’s laboratory in Moscow.

We will use the autoantibody detection kit ELI-Viscero-Test-24, “Evaluation of general state of the organism”, and: ELI-Neuro-Test-12, “evaluation of the state of the nervous system”, Immunculus, Moscow, Russia). (As alluded to above there is little data on what specific anatomical/cellular structures are affected in ME/CFS, thus the choice of autoantibodies to be searched for in this study is somewhat arbitrary. However, there are clear indications that the nervous system is affected in ME/CFS. Therefore the test “evaluation of the state of the nervous system” is included in addition to the more general evaluation.)

Calculation of results is described in the document “ELI-Viscero-Kit-Instruction” on page 10 (see attached). In short, the relative reactivity of sample serum (for each individual antibody measured) as compared to standard serum (control) is measured, and a total relative reactivity level for an individual is calculated by adding all the individual levels and dividing by the number of antibodies analyzed in a given test group.

For our main objective, the sum total for the percentage deviation from the adjusted mean for each autoantibody for each subject will be calculated. The average percentage deviation for the ME/CFS patient group and healthy subject group will be calculated. We will use the Student’s t-test to explore if there is a significant difference in the average deviation between ME/CFS patients and healthy subjects.

Costs

ELI-Viscero-Test-24, (Immunculus, Moscow, Russia) (evaluation of general state of the organism):

  • 600 USD per 10 patients

ELI-Neuro-Test-12 (Immunculus, Moscow, Russia)( “evaluation of the state of the nervous system”)

(Since the two sets of tests are in part overlapping, dsDNA and b-Glycoprotein I will be replaced by b-Endorphine and m-Opiate receptors in ELI-Neuro-Test-12.)

  • 700 USD per 30 patients

Thus in total: 7500 dollars for test kits for 90 study subjects.

  • In addition there will be travel expenses: Carrying the blood samples to Moscow for free analysis at Immunculus
Investigators

Tor Lea, NMBU, Ås, and Inge Lindseth, 4M-klinikken/Balderklinikken, Oslo, and Autoimmunity Research Foundation (ARF), California, USA (principal investigators)

Katarina Lien and Per Ole Iversen, UiO

Amy Proal and Trevor Marshall, ARF

Financing

The study will be funded by Autoimmunity Research Organization, 3423 Hill Canyon Ave, Thousand Oaks, CA 91360, USA and UGESA, Rurtalstrasse 1, 52525 Heinsberg, Germany.

Reference

1 Oscar-Danilo Ortega-Hernandez and Yehuda Shoenfeld, “Infection, Vaccination, and Autoantibodies in Chronic Fatigue Syndrome, Cause or Coincidence?,” Annals of the New York Academy of Sciences 1173 (September 2009): 600–609, doi:10.1111/j.1749-6632.2009.04799.x.

2 A B Poletaev et al., “Immunophysiology versus Immunopathology: Natural Autoimmunity in Human Health and Disease,” Pathophysiology: The Official Journal of the International Society for Pathophysiology / ISP 19, no. 3 (June 2012): 221–31, doi:10.1016/j.pathophys.2012.07.003.

3 Marta Curriu et al., “Screening NK-, B- and T-Cell Phenotype and Function in Patients Suffering from Chronic Fatigue Syndrome,” Journal of Translational Medicine 11 (2013): 68, doi:10.1186/1479-5876-11-68.

4 Ekua Weba Brenu et al., “Role of Adaptive and Innate Immune Cells in Chronic Fatigue Syndrome/myalgic Encephalomyelitis,” International Immunology 26, no. 4 (April 2014): 233–42, doi:10.1093/intimm/dxt068.

5 Poletaev mfl., «Immunophysiology versus Immunopathology».

6 Roland Jonsson et al., “Autoantibodies Present before Symptom Onset in Primary Sjögren Syndrome,” JAMA: The Journal of the American Medical Association 310, no. 17 (November 6, 2013): 1854–55, doi:10.1001/jama.2013.278448.

7 E Y Padalko and X Bossuyt, “Anti-dsDNA Antibodies Associated with Acute EBV Infection in Sjögren’s Syndrome,” Annals of the Rheumatic Diseases 60, no. 10 (October 2001): 992.

8 C Y Tsai et al., “Polyclonal IgG Anti-dsDNA Antibodies Exert Cytotoxic Effect on Cultured Rat Mesangial Cells by Binding to Cell Membrane and Augmenting Apoptosis,” Scandinavian Journal of Rheumatology 22, no. 4 (1993): 162–71.

9 Rohan Willis and Silvia S Pierangeli, “Anti-β2-Glycoprotein I Antibodies,” Annals of the New York Academy of Sciences 1285 (May 2013): 44–58, doi:10.1111/nyas.12080.


Vedlegg:

Clinical Studies ELI-P Complex: Summary

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Price (Kits)

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