The Vermont CFIDS Association reports on a recent pilot study and invites contributions to enable further research:
While preliminary, the results from this University of Vermont study is the first evidence for the potential existence of autoantibodies in ME/CFS, indicative of the presence of an inflammatory response which could in part mediate some of the symptoms observed in this disease. Future studies are planned to further investigate this new finding.
This pilot project was funded by the New Jersey ME/CFS Association. The Vermont-based organization, ImmuneDysfunction.org, is soliciting funds to expand the study. Contact them for additional details: admin@immunedysfunction.org
Estimated cost: $27,000
Pilot study introduction:
- Chronic Fatigue Syndrome (CSF), recognized as Myalgic Encephalomyelitis (ME) by the World Health Organization, is a devastating neuroimmune disease that affects over two million Americans [1,2].
- CFS/ME symptoms include a profound post-exertion fatigue and a prolonged metabolic recovery period in response to even modest physical or mental activity. Muscle pain, cognitive difficulties, somatosensory hypersensitivity, poor temperature control, and unrefreshing sleep are associated with the disease.
- Research shows that CFS/ME stems from an autoimmune reaction to a viral, bacterial, or environmental trigger [3].
- The presence of anti-citrullinated protein antibodies in rheumatoid arthritis (RA)[4.5], another autoimmune disease, suggests the possibility that abnormal citrullination of a protein may underlie the physical and cognitive impairments of CFS/ME patients. Citrullination is an enzymatic process that converts arginine into citrulline, which could trigger an autoimmune reaction.
Hypothesis
It is possible that some system protein in CFS/ME patients becomes abnormally
citrullinated in response to a viral infection (or some other foreign agent).
The immune system, recognizing the citrullinated protein(s) as a foreign object, will generate an antibody (an ‘autoantibody’). The autoantibody will attack the modified protein, which results in compromised protein function and tissue inflammation. No previous studies have reported the presence of an anti-citrullinated protein antibody (ACPA) in CFS/ME.
We tested the hypothesis that plasma from blood drawn from CFS/ME patients contains an ACPA that is not present in healthy subjects.
Methods
We used an ELISA based system in which a probe, a cyclic citrullinated peptide (CCP),
binds to an anti-citrullinated protein antibody (ACPA), if present. The system, routinely
employed in RA diagnosis, was used to measure the titers of two anti-CCP antibodies,
IgG1 and IgG4. A colorimetric assay employed a secondary antibody, pre-conjugated to
an enzyme that catalyzes a chromogenic substrate.
Serum from 25 adult patients were analyzed and compared to results from 25 healthy
age- and sex-matched control subjects. Blood plasma samples were obtained from a
national repository (the Solve CFS BioBank), administered by the Solve CFS/ME Initiative
(a national organization dedicated to CFS/ME education and research). Subject
information was kept confidential.
Fifty 100 microliter aliquots were coded with a unique identifier and sent to Dr. Rincon, who conducted the blinded analysis. The aliquots were examined for anti-CCP antibody levels (IgG1 and IgG4) using a QUANTA Lite CCP 3.1 IgG/IgA ELISA clinical diagnostic kit, i.e. the same one (with some modifications) as that used to detect the presence of anti-CCP antibodies in the serum of patients with RA [20].
Results
- Plasma samples from 7 of 25 CFS/ME subjects tested positive for anti-CCP antibody
- Plasma samples from 2 of 25 control subjects tested positive for anti-CCP
antibody. - Of the 25 CFS/ME samples, 2 tested positive for IgG1-anti-CCP antibody, 4 for
IgG4-anti-CCP antibody, and one for both. - Of the 25 control samples, 2 tested positive for IgG4-anti-CCP but none for IgG1-antiCCP.
- It is possible that the two who tested positive had RA [6] .
Discussion
An anti-citrullinated protein antibody (ACPA) was detected in roughly a third of the ME/CFS patients, advancing the possibility that citrullination of a protein or family of
proteins may underlay the physical and cognitive disabilities in some CFS/ME patients.
However, this pilot study was limited by the small number of samples tested (50),
requiring additional tests with larger sample sets (e.g., 250 or 500 samples) for
confirmation and validation. If validated, our results would strongly suggest that a
significant subset of CFS/ME patients (perhaps up to a third of the ~2 million Americans
with the disease) may have an ACPA-based autoimmunity that causes the disease
phenotype.
CFS/ME is a systems-wide neuroimmune disease with metabolic abnormalities that lead
to profound fatigue. The precise cause of CFS/ME is not known, but autoimmunity (with
genetic susceptibility) is strongly indicated [7]). Gene signatures studies report differences between CFS/ME and healthy subjects, with sensory, adrenergic and immune system receptor expression level elevated in a majority of CFS/ME patients and alpha 2A receptor expression level reduced in the remainder [8,9].
Immune system disturbances have been correlated with altered cytokine number, CD4 cell count, and RNAse L levels [10]. Viral infections such as cytomegalovirus (CMV), herpes virus HHV- 6, and particularly Epstein-Barr virus (EBV) have often been associated with CFS/ME [7].
Anti-viral agents such as valacyclovir [11] or valganciclovir [12] have been reported to
benefit some patients who are chronically infected with EBV, CMV, or other herpes
viruses.
In light of the effect CFS/ME on the brain, central nervous system, and muscle function,
our results suggest the possibility that an ACPA targets systemic proteins serving these
systems. Proteins of the vasculature and/or mitochondria are prime candidates. It is
reasonable to speculate that signaling pathways leading to citrullination of the proteins
are triggered by traumatic stress, such as infection with EBV, assisted by a genetic
vulnerability.
Target proteins
Based on literature reports [13-19], likely target proteins may be:
- Na/K-ATPase – a plasma membrane ion transporter critical for maintaining a proper electrochemical gradient in the cell.
- Endothelial or neuronal nitric oxide synthase and/or cystathionine γ-lyase (an enzyme that produces hydrogen sulfide) – both regulators of blood flow in human arteries.
- ATP/ADP translocase – a component of mitochondrial membranes and vital to maintaining a proper ATP level in the cell.
If, for example, a citrullinated Na-K pump slows down due to the action of an auto-antibody, the K+ gradient will decrease, thereby depolarizing a vascular muscle cell (due to the high membrane permeability to K+). Consequently, voltage sensitive calcium channels will open, allowing Ca++ to enter, thus causing increased muscle tension. The resulting vasoconstriction could reduce blood flow to neurons and to skeletal muscles, leading to the symptoms of CFS/ME.
In fact, disruption of any of the systemic proteins shown here (via an inflammatory autoimmune reaction) could produce symptoms of cognitive dysfunction (brain fog), disturbed sleep, exaggerated sensory modalities – and profound, sustained fatigue after physical or mental activity, all of which are hallmarks of the disease.
Conclusions
We plan to repeat this preliminary experiment using a much larger sample set. If our
results are corroborated, we will conduct future experiments designed to isolate,
identify, and characterize the putative citrullinated protein(s) – initially focusing on the
candidates above.
Our results, although preliminary, suggest the possibility that specific isotypes of anticitrullinated protein antibodies may be applied to blunt the disease pathology in specific subsets of CFS/ME patients. For example, Dr. Rincon has shown recently that
tocilizumab (a commercially available biological drug that blocks the IL-6 receptor)
selectively reduces the IgG4 type, but not the IgG1 type, of anti-CCP antibodies in RA
patients [20].
Acknowledgements
This pilot study was funded by the New Jersey Myalgic Encephalomyelitis/Chronic
Fatigue Syndrome Association. Thanks to Dr. Ken Friedman (NJME/CFSA) and Dr.
Zaher Nahle (Solve ME/CFS Initiative: www.SolveCFS.org)
References
1. Carruthers BMJ, A.K.; van de Sande, M.I.: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Clinical Working Case Definition, Diagnostic and Treatment Protocols. Journal of Chronic Fatigue Syndrome 2003, 2(1).
2. Bierl C, Nisenbaum R, Hoaglin DC, Randall B, Jones AB, Unger ER, Reeves WC: Regional distribution of fatiguing illnesses in the United States: a pilot study. Popul Health Metr 2004, 2(1):1.
3. Carruthers BMvdS, M.I.: Myalgic encephalohyeltitis – Adult & paediatric: International concensus primer for medical practitioners. In.: Carruthers& van de Sande; 2012.
4. Willemze A, Trouw LA, Toes RE, Huizinga TW: The influence of ACPA status and characteristics on the course of RA. Nat Rev Rheumatol 2012, 8(3):144-152.
5. Meyer O: Anti-citrullinated peptide/protein antibodies and structural prognosis of rheumatoid arthritis: quantity versus quality. The Journal of rheumatology 2012, 39(4):675-676.
6. Wang W, Li J: Identification of natural bispecific antibodies against cyclic citrullinated peptide and immunoglobulin G in rheumatoid arthritis. PLoS ONE 2011, 6(1):e16527.
7. Fluge O, Bruland O, Risa K, Storstein A, Kristoffersen EK, Sapkota D, Naess H, Dahl O, Nyland H, Mella O: Benefit from B-lymphocyte depletion using the anti-CD20 antibody rituximab in chronic fatigue syndrome. A double-blind and placebo-controlled study. PLoS One 2011, 6(10):e26358.
8. Whistler T, Jones JF, Unger ER, Vernon SD: Exercise responsive genes measured in peripheral blood of women with chronic fatigue syndrome and matched control subjects. BMC Physiol 2005, 5(1):5.
9. Light AR, Bateman L, Jo D, Hughen RW, Vanhaitsma TA, White AT, Light KC: Gene expression alterations at baseline and following moderate exercise in patients with Chronic Fatigue Syndrome and Fibromyalgia Syndrome. J Intern Med 2012, 271(1):64-81.
10. Chuchuen O, Henderson MH, Sykes C, Kim MS, Kashuba AD, Katz DF: Quantitative Analysis of Microbicide Concentrations in Fluids, Gels and Tissues Using Confocal Raman Spectroscopy. PLoS One 2013, 8(12):e85124.
11. Lerner AM, Beqaj SH, Deeter RG, Fitzgerald JT: Valacyclovir treatment in Epstein-Barr virus subset chronic fatigue syndrome: thirty-six months follow-up. In vivo 2007, 21(5):707-713.
12. Montoya JG, Neely MN, Gupta S, Lunn MR, Loomis KS, Pritchett JC, Polsky B, Medveczky PG: Antiviral therapy of two patients with chromosomally-integrated human herpesvirus-6A presenting with cognitive dysfunction. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 2012, 55(1):40-45.
13. Fulle S, Belia S, Vecchiet J, Morabito C, Vecchiet L, Fano G: Modification of the functional capacity of sarcoplasmic reticulum membranes in patients suffering from chronic fatigue syndrome. Neuromuscular disorders : NMD 2003, 13(6):479-484.
14. Pall ML: Nitric oxide and the etiology of chronic fatigue syndrome: Giving credit where credit is due. jmehy 2005.
15. Ali MY, Ping CY, Mok YY, Ling L, Whiteman M, Bhatia M, Moore PK: Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? British Journal of Pharmacology 2006, 149:625.
16. Lemle MD: Hypothesis: Chronic Fatigue Syndrome, Mitochondrial Hypo-function, and Hydrogen Sulfide. In: Original manuscript. 2007.
17. Lemle MD: Hypothesis: chronic fatigue syndrome is caused by dysregulation of hydrogen sulfide metabolism. Medical hypotheses 2009, 72(1):108-109.
18. Myhill S, Booth NE, McLaren-Howard J: Chronic fatigue syndrome and mitochondrial dysfunction. Int J Clin Exp Med 2009, 2009 (2):1.
19. Booth NE, Myhill S, McLaren-Howard J: Mitochondrial dysfunction and the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Int J Clin Exp Med 2012, 5(3):208.
20. Carbone G, Wilson A, Diehl SA, Bunn J, Cooper SM, Rincon M: Interleukin-6 receptor blockade selectively reduces IL-21 production by CD4 T cells and IgG4 autoantibodies in rheumatoid arthritis. International journal of biological sciences 2013, 9(3):279-288.
Results of pilot study: “Identification of anti-citrullinated protein antibodies in CFS, by Mercedes Rincon, George Webb, Terence Naumann, David Maughan
