Authors: H. Moldofskv, F.A. Lue, J. Dickstein, L. Poplonski, C.G. Jiang, R. Gorczynski;
University of Toronto Centre for Sleep and Chronobiology, Toronto, Ontario, Canada.
Chronic fatigue syndrome (CFS) patients have unrefreshing sleep and show disturbed sleep physiology. According to the theory of chronobiological disturbances in CFS¹, we hypothesized that CFS patients would show altered circadian sleep/wake-related neuroendocrine and immune functions.
Method: After overnight acclimatization to the sleep lab, thirty-four 10 ml. serial venous blood samples were collected from eleven CFS (8 f & 3 m, mean age 37.3 yrs.) and two normal comparison groups, H_Sym (healthy with symptoms of fatigue, sleep difficulties, headache, and backache; 5 f & 1 m, mean age 30.3 yrs.) and H_ASym (healthy with absence of symptoms: 4 f &1 m, mean age 30.2 yrs.). Starting at 0700 h, hourly samples were taken until 2300 h, then half-hourly during sleep until 0700 h and then at 0800 h. Polysomnographic monitoring of sleep, symptoms and mood were conducted on both days. NK-cell activity was carried out using a Cr51; release bioassay. The proportions of NK-cells and T cells subsets of peripheral blood mononuclear cells (PBM) were analyzed by immuno-fluorescence technique with flow cytometry; PBM were stained with cell surface markers for natural killer (NK) cells (CD3-, CD16+, CD56+) and T-cells (CD3+, CD16-, CD56-). The plasma was assayed for cortisol, human growth hormone (hGH), and prolactin by immunoradiometric assays. Bioassays were z-transformed for removal of between-subjects' effects. Samples were grouped into hourly means and analyzed using analysis of variance. Significant differences were examined by SNK contrast test. Similar analyses were performed for sleep stages. Cosinor analysis was applied to selected circadian variables.
Results: All groups showed NK cell decline in both proportion (F[23,420]=19.0, < .000l) and activity (F[23,413]=6.2, p < .0001) during the night. Similarly, T cell proportion increases during the night in all groups (F[23,420]=6.7, p < .000l). However, NK proportion showed a different diurnal pattern for CFS than normals (F[46,420]=l.5,p < .03). Overall the NK proportion was lower in CFS and H_Sym, than H_ASym F[2,l9]=4.6, p < .02: CFS 7.5% & H_Sym 7.7% vs H_Asym=l1.1% ). Although all groups showed the well-recognized diurnal pattern in cortisol (F[23,421]=24.6, p < .000l), CFS showed a different diurnal pattern than H_ASym and H_Sym (F[46,421]=1.4, p < .05), with phase advance on Cosinor analysis. Similarly, CFS showed a different diurnal pattern of prolactin than H_Sym & H_ASym (F[46,421]=l.9, p < .0005), but CFS and H_ASym showed lower overall prolactin than H_Sym (F[2,87]=3.5, p<.05, CFS 18.8, H_ASym 18.2 vs H_Sym 25.2). All groups showed the sleep-related increase in hGH secretion (F[6,87]=5.0, p < .0002). CFS showed higher alpha non-REM ratings (0-4) than normals (CFS 2.5 vs. H_Sym 1.7 and H_ASym 1.5) and reported more pre-sleep fatigue (p < .00l), sleepiness (p < .004), and pain (p < .00l) than H_Sym & H_ASym.
Conclusions: In comparison to normal symptomatic and asymptomatic subjects, CFS patients show altered diurnal patterns in cortisol, prolactin, and NK cells that accompany the alpha EEG sleep disorder, and daytime fatigue, sleepiness and pain. These findings are consistent with the theory of chronobiological disturbances in CFS¹.
¹ Moldofsky H. Adv. in Neuroimmunology, 5:39-56, 1995.
Research supported by a grant from The Linbury Trust Foundation.
Authors: Konstantin Konstantinov, Mark Daniels, Li Yang, Larry Gerace and Eng M. Tan. Autoimmune Disease Center and Department of Cell Biology, The Scripps Research Institute, La Jolla, California.
Objective: In previous studies (J Clin. Invest. 98:1888-1896, 1996; Arthritis Rheum. 40: 295-305, 1997), it was found that patients with chronic fatigue syndrome (CFS) had autoantibodies to a relatively insoluble cellular antigen localized at the nuclear envelope called lamin B1. Autoantibodies were generally of low titer and it was important to develop a quantitative or semi-quantitative assay to distinguish between the low positive signals given by CFS autoantibodies from background.
Methods: A cDNA clone encoding human lamin B1 was isolated from a cDNA expression library. Recombinant protein was expressed from this cDNA clone and used in immunoblotting assays. Further refinement of the methodology was made using molecular constructs of the cDNA clone where the middle region of lamin B1 consisting of coiled-coil rod domain structure was deleted and two fragments encoding primarily the N-terminal and the C-terminal domains were made. These new constructs were also expressed as recombinant proteins and used in immunoassays as antigens.
Results: Full-length recombinant human lamin B1 was used as the antigen in Western blotting and positive reactions were confirmed in over 50% of sera from patients with chronic fatigue syndrome, as had been previously reported. However, weakly positive signals were also observed in normal human sera. Many blocking reagents were used in the Western blotting assay in order to reduce or eliminate the weakly positive signals with normal sera and it was found that the best conditions were with 1% PVP-40 in PBS containing 0.05% Tween-20 and blocking for 1.5 hours at room temperature. The observation that normal human sera were also reactive with lamin B1, but to a lesser degree than CFS sera suggested that there might be an epitope on lamin B1 that was specific for CFS and probably not recognized by immunoglobulin from normal sera. Therefore, recombinant proteins from the N-terminal and C-terminal constructs were used in the slot blot technique which avoids electrophoresis and electrotransfer and simultaneous comparisons of reactivity with the N-terminal fragment (N), the full-length protein (F), and the C-terminal (C) were made. CFS sera reacted with greater intensity with N, and with much lower intensity with F and C, whereas sera from patients with lupus and other autoimmune diseases reacted with greater intensity with F and C, but with much lower intensity with N. Normal human sera were either completely negative, or reacting weakly with F and C.
Conclusion: CFS patients have autoantibody responses which target epitope or epitopes in the N-terminal region of lamin B1. An immuno-slot blot assay comparing reactivities between the N-terminal fragment and full-length lamin B1 appears to be the best discriminant to identify this auto antibody.
Authors: Aristo Vojdani, Paul Choppa, Cathy Tagle, Lilia Magtoto, and Charles Lapp; Immunosciences Lab., Inc. and the Hunter-Hopkins Center, Beverly Hills, CA and Charlotte, NC.
Objective: A prominent feature of chronic fatigue syndrome (CFS) is a disordered immune system. Recent evidence indicates that induction of apoptosis might be mediated in a dysregulated immune system by the upregulation of growth inhibitory cytokines.¹ Therefore, the purpose of this study was to evaluate the apoptotic cell population, interferon-alpha (IFN-alpha) and the IFN-induced protein kinase RNA (PKR) gene transcripts in peripheral blood lymphocytes (PBL) of CFS individuals, as compared to healthy controls.
Methods: PBL were isolated from CFS (n=29) and healthy control individuals (n=l5) and subjected to quantitative analysis of apoptotic cell population and cell cycle progression by flow cytometry. Quantitative competitive polymerase chain reaction (Q/C PCR) and Western blot analysis were used to assess the levels of PKR mRNA and protein in control and CFS individuals. In addition, circulating
IFN-alpha was measured by ELISA assay.
Results: Increased apoptotic cell population was observed in CFS individuals, as compared to healthy controls (26.6 + 12.9% and 9.9 + 4.2%, respectively). The increased apoptotic subpopulation in CFS individuals was accompanied by an abnormal cell arrest in the S phase and the G2/M boundary of the cell cycle as compared to the control group (8.6 + 1.2 to 22.8 + 2.4 and 3.6 + 0.82 to 24.3 + 3.4, respectively). In addition, CFS individuals exhibited enhanced PKR mRNA and protein levels (mean basal level 3538 + 1050 and 2.7+0.26, respectively), as compared to healthy controls (mean basal level 562 + 162 and 0.89 +0.18, respectively). In 50% of the CFS samples (n=29) treated with 2-aminopurine (2-AP) (a potent inhibitor of PKR), the apoptotic population was reduced by > 50%. Induction of PKR in CFS patients associated with the level of INF-alpha indicates activation of the interferon cascade.
Conclusions: Enhancement of the interferon-induced enzyme PKR at the mRNA and protein level, along with elevated apoptotic cell population in CFS patients, may contribute to the pathogenesis and the fatigue symptomatology associated with CFS.
1. Vojdani, A; Ghoneum, M; Choppa, PC; Magtoto, L; Lapp, CW; Elevated Apoptotic Cell Population in Patients with Chronic Fatigue Syndrome: The Pivotal Role of Protein Kinase RNA. J. Internal Medicine 242:465-478, 1997.
Authors: S. Wagner, M.S.; L. Helder, Ph.D.; N. Klimas, M.D.; M. Antoni, Ph.D.; R. Keller, M.D.
Objective: The purpose of the present study was to investigate the relationship between immunologic status and physical symptoms in Chronic Fatigue Syndrome (CFS) patients.
Methods: Twenty-seven patients diagnosed with CFS by their physician participated in a study of stress management and CFS. Subjects were 85.2% female and 14.8% male and ranged in age from 27 to 73 (M= 46.1). Participants completed a self-report questionnaire including selected subscales of the Sickness Impact Profile (yielding a Physical Impairment Score and a Total Impairment Score [Psychosocial Impairment + Physical impairment]), the Cognitive Difficulties Scale, and frequency and severity of CFS-related physical symptoms. Blood was also drawn at the time of questionnaire completion. Cellular immune markers measured included both number and percent of T-helper/inducer cells (CD3+CD4+), T-cytotoxic/suppressor cells (CD3+CD8+), activated T-lymphocytes (CD26+CD2+CD3+), activated T cytotoxic/suppressor cells (CD38+HLA-DR+8+), and CD4/CD8 ratio.
Results: Spearman's correlation coefficients revealed significant associations between a number of immunologic measures and severity of illness. Specifically, a lower percentage of
T-suppressor/cytotoxic cells was associated with greater number of cognitive difficulties (r = -.61***), SIP Total (r = -.47**) and Physical Impairment Score (r = -.39**), frequency (r = -.43**) and severity of memory problems (r = -.43**), frequency of headaches (r = -.70***), and severity of fatigue (r = -.39**). Lower number of T-suppressor/cytotoxic cells was also associated with greater headache frequency
(r = -.54***). On the other hand, a higher number of T-helper/inducer cells was associated with greater frequency of tender lymph nodes (r = .46**); a higher percentage of T-helper/inducer cells was associated with greater severity of concentration/memory difficulties (r = .40**) and headaches (r = .39*), and a greater frequency of memory problems (r = .37*) and tender lymph nodes (r = .35*). Similarly, a higher CD4/CD8 ratio was associated with greater severity (r = .43**) and frequency of memory problems
(r = .40**), frequency of headaches (r = .54***), degree of cognitive difficulties (r = .55***) and SIP Physical (r = .40**) and Total Impairment (r = .40**). Higher activated T-cell number was associated with greater frequency of tender lymph nodes (r = .52***) and cognitive difficulties (r = .34*). Greater number of activated cytotoxic/suppressor T-cells was associated with greater severity of tender lymph nodes (r = .45**), fatigue (r = .34*), and ~eep problems (r = .34*). Finally, a higher percentage of activated cytotoxic/suppressor T-cells was associated with greater severity of fatigue (r = .35*) and tender lymph nodes (r = .39*).
Conclusion: These findings suggest that the degree of cellular immune activation is associated with the severity of CFS-related physical symptoms, cognitive complaints, and perceived impairment secondary to CFS. Specifically, elevations in T-helper/inducer cells, activated T-cells, activated cytotoxic/suppressor T-cells, and CD4/CD8 ratio are associated with greater disease severity. Furthermore, reductions in T-suppressor/cytotoxic cells also appear related to greater severity of CFS-related physical symptoms and illness burden, suggesting greater symptoms are associated with lower availability of regulatory T-cells.
Authors: O. Zhang, B.H. Natelson, X.D. Zhou, T. Denny, J.E. Ottenweller, W.C. Gause
Objective: This study was to evaluate the hypothesis that CFS is associated with a disregulated immune system marked by increased Type II T-cell responses and cytokines.
Methods: By using multivariate mixed-effects models, we compared multiple immunological indicators of Gulf veterans (n=43) and civilians (n=68) who fulfilled published case definitions for CFS to their respective healthy controls (34 Gulf veterans and 53 civilians). Dependent variables included cell sorter based white blood cell counts (total WBC, total lymphocytes, NK-cells, total B-cells, total T-cells, CD4+ T-cells, CD8+ T-cells, and the following activation markers: CD4+CD45RO+, CD4+CD45RA+, CD8+CD11b-, CD8+CD38+, CD8+HLA-DR+, and CD8+CD28+) as well as IL-2, IL-4, IL-6, IL-10,
IL-12, IFN-gamma and TNF-alpha assayed by RT-PCR.
Results: No differences in any variable were found for the civilian group. In contrast, statistically significant differences were found in a number of immunological variables between Gulf vets with CFS and healthy Gulf vet controls, and between the former and the civilian groups. Specifically, CFS vets had higher numbers of total T-cells and of T-helper cells than both Gulf and civilian healthy controls. CFS vets also had higher percentages of total T-cells and of T-helper cells and a lower percentage of NK-cells than Gulf vet controls (these were not different from healthy civilians). However, cell surface marker data in CFS vets never fell outside the laboratory's general normative range. In addition, CFS vets had higher levels of IL-2, IL-10, IFN-gamma, and TNF-alpha than vet controls.
Conclusions: The data provided support for the immune dysfunction hypothesis in the CFS veteran group, but not in the CFS civilians. However, instead of having Type II responses, the CFS veterans showed a general upregulation of cytokines and T cells and a decreased percentage of NK-cells. One interpretation of the immunological differences exhibited by CFS Gulf vets is that immune dysregulation plays a role in the genesis of this epidemic presentation of CFS. However, differences between the veteran groups in terms of sleep, chronic stress, and level of activity also could contribute to produce the differences.