CDC: Evaluation of autoantibodies in CFS

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> [Original Message]

> From: Dr. Marc- Fluks <fluks@...>

> <CO-CURE@...>

> Date: 5/27/2005 12:58:10 PM

> Subject: [CO-CURE] RES,NOT: Evaluation of autoantibodies in CFS

>

> Source: Journal of Autoimmune Diseases

> Vol. 2:5

> Date: May 25, 2005

> URL: http://www.jautoimdis.com/content/2/1/5

>

>

> Evaluation of Autoantibodies to Common and Neuronal Cell Antigens in

> Chronic Fatigue Syndrome

> --------------------------------------------------------------------

> Suzanne D Vernon* (svernon@...)

> C Reeves (wcr1@...)

>

> Division of Viral and Rickettsial Diseases, National Center for Infectious

> Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

> 30333, United States

> * Corresponding author

>

> Submission date 19 Apr 2005

> Acceptance date 25 May 2005

> Publication date 25 May 2005

>

>

> Key Words: chronic fatigue syndrome, CFS, antibodies, autoantibodies

>

>

> Abstract

>

> People with chronic fatigue syndrome (CFS) suffer from multiple symptoms

> including fatigue, impaired memory and concentration, unrefreshing sleep

> and musculoskeletal pain. The exact causes of CFS are not known, but the

> symptom complex resembles that of several diseases that affect the immune

> system and autoantibodies may provide clues to the various etiologies of

> CFS. We used ELISA, immunoblot and commercially available assays to test

> serum from subjects enrolled in a physician-based surveillance study

> conducted in Atlanta, Georgia and a population-based study in Wichita,

> Kansas for a number of common autoantibodies and antibodies to neuron

> specific antigens. Subsets of those with CFS had higher rates of

> antibodies to microtubule-associated protein 2 (MAP2) (p = 0.03) and ssDNA

> (p = 0.04). There was no evidence of higher rates for several common

> nuclear and cellular antigens in people with CFS. Autoantibodies to

> specific host cell antigens may be a useful approach for identifying

> subsets of people with CFS, identify biomarkers, and provide clues to CFS

> etiologies.

>

>

> Background

>

> Chronic fatigue syndrome (CFS) is defined as persistent or relapsing

> fatigue that has occurred for at least 6 months, is not alleviated by

> rest, and causes substantial reduction in activities. The fatigue cannot

> be explained by medical or psychiatric conditions and must be accompanied

> by at least 4 of 8 specified symptoms (unusual post exertional fatigue,

> impaired memory or concentration, unrefreshing sleep, headaches, muscle

> pain, joint pain, sore throat, and tender cervical nodes) [1]. There is

> considerable discrepancy in results between studies from different

> institutions; so as yet, there are no characteristic signs or laboratory

> markers of CFS and its pathophysiology has not been elucidated [2].

>

>

> This lack of diagnostic signs or laboratory markers notwithstanding, many

> manifestations of CFS resemble those of musculoskeletal and infectious

> diseases [3]. In large part, the illnesses caused by these diseases

> reflect immune system activation and there is evidence for immune system

> dysfunction in some cases of CFS. In particular, antinuclear antibodies

> (ANA) and other common autoantibodies have been evaluated in people with

> CFS: unfortunately, with variable results. For example, one study found

> that 52% of tertiary care- CFS referral-patients had antibodies to nuclear

> envelope antigens [4] while another study found the same ANA antibody

> rates in both CFS and controls [5]. Recently, investigators reported that

> antibodies to the human muscarinic cholinergic receptor 1 may provide a

> biologic explanation for the cognitive impairment observed in people with

> CFS [6]. This lack of consensus between studies may in large-part reflect

> recruitment bias associated with studies of persons enrolled from tertiary

> referral clinics combined with imprecise evaluation of the illness and

> inadequate or inappropriate control populations.

>

> We had the opportunity to measure the associations of common

> autoantibodies and autoantibodies to neuronal cell antigens and CFS in two

> case control studies; one of primary care patients with CFS who were

> identified by a physician surveillance network; the other a study of

> people with CFS identified from the community. The physician surveillance

> study was conducted 1988 through 1993 in Atlanta, Georgia [7] and the

> community study 1997 through 2000 and identified subjects with CFS from

> the general population of Wichita, Kansas [8].

>

> Both studies rigorously classified people as CFS and controls in both

> studies were enrolled to represent the general population and matched to

> cases by sex, race, and age [9, 10]. The hypothesis of the present study

> is that the appearance of cell-specific autoimmune antibodies may define

> subsets of CFS and give clues to the etiology and pathogenesis and may

> help to explain the neurocognitive symptoms experienced by CFS patients.

> Secondarily, we wished to evaluate the extent to which patients with CFS

> who were receiving primary medical care treatment for CFS were similar to

> people with CFS in the community.

>

>

> Methods

>

> Study Subjects and samples

>

> Both studies adhered to human experimentation guidelines of the U.S.

> Department of Health and Human Services and the Helsinki Declaration. The

> Centers for Disease Control and Prevention (CDC) Institutional Review

> Board approved study protocols. All participants were volunteers who gave

> informed consent.

>

> Physician surveillance study. Between 1988 and 1993, the CDC conducted a

> physician surveillance survey for CFS in primary care patients from Reno,

> Nevada, Wichita, Kansas, Grand Rapids, Michigan, and Atlanta, Georgia [7].

> Patients were classified as CFS according to the 1988 case definition

> [11]. In 1992, we conducted a case control study of CFS patients and

> controls in Atlanta by recruiting patients from physician surveillance and

> sex, race, age matched non fatigued controls identified in the general

> Atlanta population [9, 10]. The case control study classified patients as

> CFS according to the study collected information concerning several risk

> factors and blood to measure associations between CFS and laboratory

> markers. The present study used remaining archived serum samples from 22

> CFS patients and 34 age and sex matched controls. All CFS patients met

> criteria of the current CFS research case definition [1]

>

> Population study participants. Between 1997 and 2000, CDC conducted

> surveillance of CFS in the general population of Wichita, Kansas [8].

> Briefly, the study involved random digit dial surveys to identify people

> with CFS-like illness and clinically evaluated and classified them

> according to criteria of the 1994 CFS research case definition [1]. Only

> 16% of those identified with CFS had been diagnosed or treated for CFS by

> a physician [12]. The present study used archived serum samples from 37

> subjects with CFS and a 57 non-fatigued control subjects.

>

> Blood samples. Both the physician surveillance and population study

> collected blood in BD Vacutainer Serum tubes. The samples were shipped by

> overnight courier to CDC where they were dispensed into 0.5 ml aliquots

> and stored at -80° C until testing.

>

>

> Reagents and Assays

>

> Commercially available kits were used for antibodies to ubiquitous nuclear

> and cellular autoantigens including dsDNA, ssDNA, Sm, U1-RNP, SS-A/Ro,

> SS-B/La, Scl-70, and Centromere. Immunoassays were purchased from Helix

> Diagnostics (West Sacramento, CA) and reagents for western blots were

> purchased from Diagnostic Products Corporation (Los Angeles, CA). Purified

> Histone H3, and Histone H4 were purchased from Sigma (St. Louis, MO). and

> used in ELISA assays that were developed at Scripps. Conventional

> immunofluorescent antinuclear antibodies and rheumatoid factor tests were

> performed as described previously [13]. Preparations of

> microtubule-associated protein 2 (MAP2) and neurofilament triplet (NFT)

> proteins were purchased from Sigma (St. Louis, MO). The commercially

> available ELISA assays were performed according to the manufacturers

> instructions. The ELISA and western blot assays for the neuronal antigens

> were developed at Scripps and were performed as previously described [14].

>

>

> Statistical Analysis

>

> Because the subjects are derived from studies that are distinct in design

> and geographic location, each study was analyzed separately. The

> distribution of autoantibodies between CFS and non-fatigued controls was

> compared by Fisher exact probability test. To derive an estimate of

> confidence, stratified groups were compared by the non-parametric chi

> square test. To determine associations, subjects were stratified by sex,

> age, and CFS for all MAP2, NFT and ssDNA. The association of autoantibodes

> in CFS subjects was compared by grouping by sex, age, age at illness

> onset, and duration of illness. CFS subjects were stratified by sex, age

> (<40 years, 40-40 years, >50 years), onset type (gradual versus sudden)

> and duration of illness (<5 years, >5 years) to determine whether an

> association with autoantibodies existed.

>

>

> Results

>

> Although women predominated in both study groups other demographic and

> clinical characteristics differed and reflected basic differences between

> patients with CFS who obtain medical care and those in the general

> population (most of whom have not seen a physician) (Table 1). Of note,

> CFS cases from physician surveillance were somewhat younger than those

> identified in the population (mean 39 and 46 years, respectively) and

> controls were similarly different: those recruited in the physician study

> had been ill about half as long as those in the community (69 and 128

> months, respectively) and were more likely to report sudden onset CFS

> (36.4%) than those in the general population with CFS (13.5%).

>

> A few CFS subjects in the physician surveillance study aged 18-29 years

> had antibodies to ssDNA when compared to the same age non-fatigued control

> group. The mean value for the 3 CFS subjects was 2-fold greater then in

> the 6 non-fatigued controls (p = 0.038). Among CFS subjects, the 10 who

> reported being ill for 5 years had lower levels of autoantibodies to MAP2

> (median value of 18, range 12 - 20) compared to the 12 CFS subjects who

> have been ill for >5 years (median value of 8, range 6 to 10) (p = 0.025).

> There were no other significant findings in the physician surveillance CFS

> subjects when stratified by sex or type of illness onset.

>

> In the population-based study, there was a significant difference in the

> prevalence of autoantibodies to MAP2 between the 30 male subjects (20/30,

> 67% positive) and the 64 female subjects (19/64, 30% positive) (p=0.0006).

> Among the non-fatigued control group, 9 of 33 women (27%) and 19 of 24

> (79%) men were positive for antibodies to MAP2 (p = 0.0004). One male CFS

> subject (16%) was positive for MAP2 antibodies compared to 79% (19/24)

> male non-fatigued controls (p = 0.04). Among CFS subjects that were 40

> years of age, there was a trend for lower MAP2 antibody levels for those

> that were ill for 5 years compared to those ill for >=5 years (p=0.056).

>

>

> Discussion

>

> CFS is a complex, debilitating illness, which is characterized by at least

> 6 months of severe persistent or relapsing fatigue and a group of

> characteristic but nonspecific symptoms. Despite more than a two decades

> of extensive research, no diagnostic tests exist, and effective control

> and prevention remain elusive because the cause and pathophysiology of CFS

> remain unknown. CFS is clinically similar to several rheumatic autoimmune

> disorders that can be diagnosed and characterized by autoantibody

> profiles. For this reason, we conducted an exhaustive evaluation of 11

> ubiquitous nuclear and cellular autoantigens in addition to two neuronal

> specific antigens.

>

> The serum samples tested in this study were collected from a physician

> surveillance study conducted in Atlanta [15] and a population-based

> community study in Wichita [8]. The physician surveillance study was

> conducted over 7 years and used approximately 70% of all primary care

> physicians in Atlanta. All patients were carefully evaluated for

> unexplained unwellness and fatigue. The community study was a random digit

> dial survey of 90,000 people (25% of the Wichita, Kansas population). All

> CFS cases were medically and psychiatrically evaluated and rigorously

> classified as CFS, other unexplained unwellness, or

medically/psychiatrically

> explained unwellness. The serum evaluated in this study includes carefully

> evaluated CFS subjects, matched controls and non-fatigued controls from

> the community. Therefore, the results from this study should be applicable

> to similarly designed studies.

>

> Very few studies have evaluated the presence of autoantibodies in people

> with CFS. Those that have tested for the same autoantibodies report

> discordant results. Konstantinov et al, [4] found high rates of

> antinuclear antibodies (ANA) in CFS patients while Skowera et al, [5]

> found no difference in the rate of ANA between CFS patients and controls.

> One explanation for these discrepancies could be a technical one where

> laboratories used different reagents and methods resulting in discordant

> results. Another explanation could be that the CFS subjects were evaluated

> differently. Rigor in evaluating CFS patients and applying the case

> definition [1] is pivotal and undoubtedly accounts for much of the

> variation. The results presented also show little evidence for

> autoantibodies to ubiquitous nuclear and cellular autoantibodies.

>

> The findings of this study hint that evaluation of certain autoantibodies

> may give clues to etiology and ongoing pathology in subsets of CFS

> subjects. A few of the physician surveillance CFS cases from the youngest

> age category had autoantibodies to ssDNA. Autoantibodies to ssDNA have

> been associated with both viral and bacterial infection [16, 17].

> Interestingly, CFS subjects who describe a sudden onset to their illness

> often report flu-like illness. The fact that antibodies to ssDNA were

> detected only in this age group may reflect an immune response to

> infection commonly affecting this age group, such as infectious

> mononucleosis from Epstein Barr Virus infection.

>

> There was a higher prevalence of autoantibodies to MAP2 in the

> non-fatigued men in the community study compared to the non-fatigued

> women. The significance of this finding is not known but highlights the

> importance of carefully stratifying and controlling for factors that could

> affect interpretation of results. There did seem to be a slight

> association of MAP2 autoantibodies with duration of CFS illness. Among CFS

> subjects in both study populations, those who had been sick longer had

> higher rates of autoantibodies than those that report shorter duration of

> illness. MAP2 is a neuron specific cytoskeleton protein. Autoantibodies to

> MAP2 have been demonstrated in patients with neuropsychiatric systemic

> lupus erythematosus [14]. While no lesions or loss of central nervous

> system function is reported in people with CFS, loss of memory,

> concentration and cognitive impairment are common complaints. Future

> studies will attempt to associate assessment of these parameters with the

> presence of MAP2 autoantibodies.

>

>

> Conclusions

>

> There was no evidence of higher rates of the common autoantibodies in

> people with CFS. However, certain subsets of CFS subjects that had higher

> rates of antibodies to microtubule-associated protein 2 (MAP2) and ssDNA.

> Autoantibodies to specific host cell antigens may be a useful approach to

> stratify CFS subjects and provide clues to CFS etiologies.

>

>

> Competing interests

>

> None declared.

>

>

> Authors' contributions

>

> SDV was instrumental in the design of the experimental approach, analysis,

> presentation, discussion of these data and manuscript preparation. WCR

> contributed to the design of the experimental approach, and to the

> manuscript preparation. Both authors read and approved the final

> manuscript.

>

>

> Acknowledgments

>

> We would like to thank Dr. Eng Tan of The Scripps Research Institute for

> conducting the autoantibody assays. We would also like to thank Dr. ne

> Nisenbaum for assistance with the statistical analysis.

>

>

> References

>

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and

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about

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GP,

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> JA, Abbey S, JF, Gantz N, Minden S, Reeves WC: Prevalence and

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Steele L,

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SE,

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microtubule-associated

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>

>

> Table 1. Characteristics of subjects evaluated for autoantibodies

> -----------------------------------------------------------------

> CFS Non-Fatigued

> -----------------------------------------------------------------

> Atlanta Case Control

> Subjects (n=56) 22 34

> Female (n=52) 19 33

> Male (n=4) 3 1

> Age Group (yrs)

> 18-29 (n=9) 3 6

> 30-39 (n=18) 7 11

> 40-49 (n=23) 12 11

> 50-59 (n=6) 0 6

> Mean Age 39 years 38 years

> Mean Age Onset 35 years

> Onset type

> Sudden 8

> Gradual 14

> Mean Illness Duration 69 months

> Number of Subjects Ill

> < 5 years 10

> >5 years 12

>

> Wichita Population

> Subjects (n=94) 37 57

> Female (n=64) 31 33

> Male (n=30) 6 24

> Age Group (yrs)

> 18-29 (n=14) 1 13

> 30-39 (n=18) 8 10

> 40-49 (n=26) 13 13

> 50-69 (n=36) 15 21

> Mean Age 46 years 42 years

> Mean Age Onset 36 years

> Onset type

> Sudden 5

> Gradual 32

> Mean Illness Duration 128 months

> Number of Subjects Ill

> < 5 years 14

> >5 years 23

> -----------------------------------------------------------------

>

> --------

> © 2005 Vernon and Reeves, licensee BioMed Central Ltd.

>

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