Source: Medical Hypotheses Vol 62, #5, pp 759-765 Date: April 2004 URL: http://www.sciencedirect.com/science/journal/03069877 Chronic fatigue syndrome: intracellular immune deregulations as a possible etiology for abnormal exercise response ------------------------------------------------------------ Jo Nijs(a,b,*), Kenny De Meirleir(a,c), Mira Meeus(a), Neil R. McGregor(d,e), Patrick Englebienne(f,g) a Department of Human Physiology, Faculty of Physical Education and Physical Therapy Science, Vrije Universiteit Brussel (VUB), Brussel 1090, Belgium b Institute for Occupational and Physical Therapy, Department of Health Sciences, Hogeschool Antwerpen, Belgium c Chronic Fatigue Clinic, Vrije Universiteit Brussel (VUB), Belgium d Bio21, Institute of Biomedical Research, University of Melbourne, Parksville, Victoria 3000, Australia e Dental Clinical School, Westmead Hospital, Westmead, Australia f Department of Nuclear Medicine, Universit e Libre de Bruxelles (ULB), Belgium g RED Laboratories N. V., Zellik, Belgium * Corresponding author. Present address: Vakgroep MFYS/Sportgeneeskunde, AZ-VUB KRO gebouw - 1, Laarbeeklaan 101, 1090 Brussel, Belgium. Tel.: +32-2-477-4604; fax: +32-2-477-4607. E-mail address: jo.nijs@vub.ac.be Received 1 October 2003; accepted 9 November 2003 Summary The exacerbation of symptoms after exercise differentiates Chronic fatigue syndrome (CFS) from several other fatigue-associated disorders. Research data point to an abnormal response to exercise in patients with CFS compared to healthy sedentary controls, and to an increasing amount of evidence pointing to severe intracellular immune deregulations in CFS patients. This manuscript explores the hypothetical interactions between these two separately reported observations. First, it is explained that the deregulation of the 2-5A synthetase/RNase L pathway may be related to a channelopathy, capable of initiating both intracellular hypomagnesaemia in skeletal muscles and transient hypoglycemia. This might explain muscle weakness and the reduction of maximal oxygen uptake, as typically seen in CFS patients. Second, the activation of the protein kinase R enzyme, a characteristic feature in atleast subsets of CFS patients, might account for the observed excessive nitric oxide (NO) production in patients with CFS. Elevated NO is known to induce vasidilation, which may limit CFS patients to increase blood flow during exercise, and may even cause and enhanced postexercise hypotension. Finally, it is explored how several types of infections, frequently identified in CFS patients, fit into these hypothetical pathophysiological interactions. --------------------------------------------------------------------------------- Introduction To date, the most widely used criteria used to establish the medical diagnosis of 'Chronic fatigue syndrome (CFS) ' are those reported in 1994 by the Center for Disease Control and Prevention [1]. According to this operational definition, a CFS patient presents with severe fatigue and a number of other symptoms (myalgia, arthralgia, low-grade fever, concentration difficulties), of atleast six months duration. The symptoms are not improved by bed rest and may be aggravated by physical or mental activity [1,2]. Importantly, any active medical condition that may explain the presence of the symptoms prohibits the diagnosis of CFS. Although CFS affects both sexes and all age groups, the typical patient is a middle-class Caucasian women in her thirties. Since the pathogenesis of the illness remains to be elucidated [3], the natural history of adult patients with CFS is poor [4]. Our current understanding of the disease mechanisms of CFS points to both physical and psychological impairments [5]. Previous research has shown that patients with CFS present with an abnormal exercise response and exacerbation of symptoms after physical activity. Some of the main findings were a reduction in maximal oxygen uptake [6Ð 10], reduction in peak heart rate [9,10] and peak power output [7], earlier exhaustion [7Ð-10], and accelerated glycolysis with increased lactate production [11]. Contrary to these findings, Sargent et al. [12], Rowbottom et al. [13], and Kent-Braun et al. [14] found that the aerobic capacity of CFS patients lies within the low normal range. The highly heterogeneous nature of the CFS population and the lack of uniformity in the utilised diagnostic criteria preclude pooling of data and hence to draw firm conclusions. Still, we conclude that at least a subgroup of CFS patients present with an abnormal response to exercise. In addition, since several exercise capacity parameters (e. g. functional aerobic impairment, bodyweight adjusted peak oxygen uptake, exercise duration) correlated with activity limitations/participation restrictions [15], evidence supporting the clinical importance of impairments in cardiorespiratory fitness in CFS patients was provided. Importantly, the exacerbation of symptoms after exercise is seen only in the CFS population, and not in fatigue-associated disorders such as depression, rheumatoid arthritis, systemic lupus erythematosus, or multiple sclerosis [16]. To date, the exact cause of the abnormal exercise response in CFS remains to be revealed. Snell and colleagues [17] showed that CFS patients with evidence of a deregulated 2,5-oligoadenylate (2-5A) synthetase/RNase (ribonuclease) L pathway have a lower peak oxygen uptake than the CFS patients without the intracellular immune deregulation, suggesting a link between immunopathology and exercise capacity in CFS. This manuscript explores the hypothetical associations between exercise pathophysiology and the recent advances in immunopathology in patients with CFS. Hypotheses CFS-associated channelopathy might cause muscle weakness and hypoglycemia The deregulation of the 2-5A synthetase/RNase L pathway in subsets of CFS patients has been reported at length in the scientific literature [18-23]. Both elastases and calpain are capable ofinitiating high molecular weight RNase L (83 kDa) proteolysis, generating two major fragments with molecular masses of 37 (a truncated low molecular weight RNase L) and 30 kDa, respectively [24]. By measuring and calculating the amount of low molecular weight protein relative to high molecular weight, we are able to quantify the deregulation of the antiviral pathway. Starting from the N-terminal end of the RNase L polypeptide, the first 330 amino-acids sequence present a high degree of homology with the ankyrin repeat motif. Proteolytic cleavage of 83 kDa RNase L generates ankyrin repeat motif-containing fragments. Ankyrins are a family of proteins that control numerous physiological processes by means of interactions with integral membrane proteins. In particular, ankyrin proteins are capable of associating with ABC transporters that they link to the cytoskeleton [25]. The RNase L-inhibitor (RLI) impairs 2-5A binding on the ankyrin domain of RNase L [22] and consequently, RLI binds ankyrin fragments released in the cells of CFS patients. RLI takes part of the ATP binding cassette (ABC) transporter superfamily. When the ankyrin fragment of RNase L is released by cleavage, it competes also with the cognate ankyrin protein ABC transporter, inducing deregulation of their proper function. Recent research revealed sequence similarity between RLI and several ABC transporters, for instance sulfonylurea receptor (SUR 1) [26]. SUR 1 is an important member of ATP-sensitive potassium channels. Impairment of SUR 1 function in cells could be postulated to lead to extreme losses of both cellular potassium and magnesium (K efflux and interdependent Mg^2+ flux). Preliminary evidence for this type of channelopathy in a subset of patients with CFS has been provided [27]. Intracellular hypomagnesaemia is a well known cause of muscle weakness, including respiratory (muscle) weakness. Consequently, the outlined channelopathy might account for the observed reduction in maximal oxygen uptake [6-10] in CFS patients. In addition, SUR1, by regulating the ATP-sensitive potassium channels, plays a key role in the regulation of glucose-induced insulin secretion [26]. Consequently, the 83 kDa RNase L cleavage induced channelopathy may cause transient hypoglycemia in subsets of patients with CFS. The above outlined mechanism might explain the observation of Snell et al., who found that CFS patients with evidence of a deregulated 2-5A synthetase/RNase L pathway have a lower peak oxygen uptake than those patients without the immune deregulation [17]. Part from initiating a channelopathy, there might be another link between muscle weakness and the deregulated 2-5A synthetase/RNase L pathway. In muscle cells, the activated RNase L system plays a role in cell differentiation (control of cell maturation, i. e. the expression of specific proteins such as alpha-actin and troponin T) [28]. Thus, a disfunctional RNase L pathway is likely to preclude physiological muscle cell maturation and hence activity [29]. This might explain part of the incapacity of CFS patients to perform a maximal effort [6-10, 15], and the muscle weakness as typically seen in CFS patients [1,2,30]. Still, part from peripheral blood mononuclear cells, the deregulation of the 2-5A synthetase RNase L pathway has not yet been documented in other cells. At this stage these pathophysiological interactions in muscle tissue of CFS patients remain purely speculative and require further investigation. The question which arises is what would cause the 83 kDa RNase L proteolysis? In theory, Mycoplasma infections are capable of doing so. Compared to healthy subjects, an increased prevalence of Mycoplasma infections has been reported in CFS patients [31Ð 33]. Mycoplasma spp. can activate monocytes, neutrofils and T-cells [34,35]. To bring about their phagocytic activity, monocytes, neutrofils and activated T-cells produce elastase [36,37]. Consequently, Mycoplasma spp. could at-least in part account for the observed proteolysis of the native 2-5A dependent RNase L into its truncated form. The latter hypothesis is supported by the observation that Mycoplasma infected CFS patients presented with more evidence of 83 kDa RNase L proteolysis, compared to non-infected CFS patients [38]. Once the 83 kDa RNase L cleavage has occurred, cell apoptosis (' programmed cell death') is initiated. Apoptosis in turn enhances the activity of pro-apoptotic and pro-inflammatory proteases [29], including both elastase and calpain, each of which is capable of high molecular weight RNase L proteolysis [24]. Thus a vicious cycle is initiated, implicating that at the time the patient's blood is investigated (i. e. when the subject visits a fatigue clinic), the organism responsible for initiating 83 kDa RNase L proteolysis might no longer be present. Future research should address the hypothetical interactions between the exercise response, 83 kDa RNase L cleavage, and Mycoplasma spp. in patients with CFS. Protein kinase R activation might prevent increased muscle blood flow during exercise in CFS patients Recently, additional evidence supporting intracellular immune deregulation in patients with CFS has been provided [39,40]. Besides triggering the 2-5A synthetase/RNase L activation, type I interferons induce the expression of the double-stranded RNA dependent protein kinase R (PKR). Activation of this enzyme, as typically seen during viral infection or cellular stress, results in a blockade of protein synthesis and consequent cell death (apoptosis). The PKR enzyme and 2-5A synthetase/RNase L system are termed the "cellular double- stranded RNA-detecting systems" that are responsible for the translational inhibition in response to (viral) infection [41]. Experimental data point to an activation of the PKR enzyme, parallel to the 83 kDa RNase L proteolysis, in subsets of CFS [39]. PKR activation leads to phosphorylation of the inhibitor of NF(nuclear factor)-kappaB (I kappaB) and consequent NF-kappaB activation, which in turn causes inducible nitric oxide synthetase (iNOS) expression [42] (Fig. 1). iNOS generates increased production of NO by monocytes/macrophages. NO, a soluble gas acting close to where it is produced, mediates important vital physiological functions such as neurotransmission, cell-mediated immune responses (strong antimicrobial and antitumour activities), and vasodilatation. Excessive and/or persistent production of NO, however, is detrimental to the body's functions [42,43]. The PKR and iNOS activation might explain the observations of Vecchiet et al. [44], who found evidence of oxidative stress in CFS patients. Elevated NO, documented in CFS patients by Kurup and Kurup [45], has been suggested as the common etiology of CFS, multiple chemical sensitivity disorders, and posttraumatic stress disorder [46]. Still, since NF-kappaB activation does not per se require activated PKR or RNase L [41,43], future research might reveal additional pathways explaining elevated NO levels in patients with CFS. NO, as a mediator of vasodilatation, is critical for basal blood flow across many organs [47]. Indeed, NO induces smooth muscle relaxation in the walls of the blood vessels [48]. Since CFS patients have been shown to have elevated NO levels at rest [46], one can expect them to present with a decreased peripheral resistance and consequent reduced blood pressure (hypotension). The latter may explain part of the abnormal response to exercise seen in subsets of patients with CFS. Indeed, NO-induced vasodilatation at rest may limit the capacity of the human body, especially in muscle tissue, to increase blood flow during exercise, consequently limiting exercise capacity in CFS patients. High amounts of NO might even explain the exacerbation of symptoms after exercise (" post-exertional malaise"), as typically seen in CFS patients [16]. In healthy subjects, a sympatically regulated vasodilatation and consequent hypotension is considered a normal response to a bout of dynamic exercise (commonly referred to as 'post-exercise hypotension'). Although this is refuted by the observations of Halliwill et al. [49], who concluded that postexercise is unlikely to be dependent on increased NO production, the high amounts of NO might enhance postexercise hypotension in CFS patients. The latter may account for a delayed recovery from exercise as typically seen in CFS patients. Another hypothetical link between increased NO availability and reduced exercise capacity is the inhibitory effect of NO on oxidative cell metabolism (and consequent reduced levels of ATP), as seen in beta-pancreatic cells in patients with diabetes [50]. Presuming that high amounts of NO impair oxidative metabolism in cardiac and skeletal muscle tissue in CFS patients, aerobic exercise capacity will be reduced. Excess NO might even explain muscle weakness and muscle fatigue in patients with CFS. Excess NO is likely to explain the observations of Fulle et al. [51] who found evidence for alterations of the opening status of ryanodine channels (Calcium channels at the sarcoplasmic reticulum regulating calcium flux). The consequent impaired calcium channels leads to a dysfunctional excitation-contraction coupling at the terminal cisternae of the sarcoplasmic reticulum in muscle cells. To date, the exact etiology of elevated NO levels in CFS patients remains to be established. It is tempting to speculate that infections, frequently associated with the illness, trigger NF-kappaB activation and consequent iNOS induction. As outlined above, an increased prevalence of Mycoplasma infections in CFS patients compared to healthy subjects has consistently been reported in the scientific literature [31-33]. Among the different Mycoplasma species studied, M. fermentans is one of the most prevalent in patients with CFS. M. fermentans produces a lipopeptide, named 2-kDa macrophage-activating lipopeptide (MALP-2), which stimulates macrophages [46]. Macrophages, activated by MALP-2, release nitric oxide [52-54]. Indeed, M. fermentans derived membrane lipoproteins are capable of rapidly activating NF-kappaB [55-58]. Thus the presence of M. fermentans on itself can explain elevated NO levels in subsets of the CFS population. Interestingly, Sorensen and colleagues found evidence of complement activation in CFS patients, and they showed that an exercise challenge further enhances complement activation [16]. M. fermentans, as well as several other species of Mycoplasma, are capable of activating the complement cascade. A M. fermantans-originating 43 kDa lipoprotein, named M161Ag, activates the human complement via the alternative pathway [59]. Apart from M. fermantans, another type of infection frequently associated with CFS is capable of triggering NF-kappaB activation and consequent iNOS induction. Indeed, Chlamydia pneumoniae (C. pneumoniae) has been identified repeatedly in blood samples of CFS patients [60,61]. Chlamydia species were even found in 2 of 12 cerebrospinal fluid samples of CFS subjects [62]. Although Fischer et al. found that C. pneumoniae does not induce significant NF-kappaB activation in human cells [63], numerous investigators have provided evidence supporting the activation of NF-kappaB by C. pneumoniae in various tissues [64-67]. As the human body recognizes C. pneumoniae, NF-kappaB activation is triggered to generate increased amounts of NO, which in turn inhibits chlamydial growth. The hypothetical interactions between the exercise response, NO concentration, blood pressure, infections with both Mycoplasma spp. and C. pneumoniae, and complement activation deserve further investigation. Furthermore, NO is synthesized in response to, and has potent antiviral activity against a number of viruses, for instance Coxsackie B virus [68], and Epstein- Barr virus [69]. Both Epstein-Barr virus and Coxsackie B virus have been suggested as cofactors in CFS pathophysiology; antibodies to Coxsackie B virus are commonly found in blood samples taken from CFS patients [70,71], while an infection of the B lymphocytes by Epstein-Barr virus has long been considered the cause of CFS [72]. Taken together, a high number of pathogens, each of which have been identified in CFS patients, are potent initiators of NF-kappaB activation and consequent iNOS induction. The latter might explain the observed high amount of NO in patients with CFS. Future research should reveal whether or not NO availability limits exercise capacity in (subsets of) the CFS population. Testing the hypotheses In order to test the above outlined hypotheses, future research should exposure a randomly allocated sample of CFS subjects to a standardized exercise protocol (monitoring cardiorespiratory parameters continuously). Prior to the exercise stress testing, a plebotomy is required in order to monitor the 83 kDa RNase L proteolysis, biochemical parameters (potassium, etc.), NO concentration, PKR activity, and NF-kappaB activity. Furthermore, it would be of interest to monitor possible changes in both health status (symptom severity and quality of life) and blood parameters up to 48 h after the bout of exercise. Although even this type of study is unlikely to provide definitive evidence, it is likely to either confirm or refute some of these hypotheses. Figure caption Figure 1 Pathophysiological mechanism, possibly explaining the hypothetical interaction between elevated NO levels and impairments in cardio respiratory fitness in CFS patients. IFN, interferon; PKR, protein kinase R; NF-kappaB, nuclear factor kappaB; iNOS, inducible nitric oxide synthetase; NO, nitric oxide; ?, hypothetical interaction. References [1] Fukuda K, Strauss SE, Hickie I, et al. The Chronic Fatigue Syndrome, a comprehensive approach to its definition and study. Ann Intern Med 1994; 121: 953-959s. [2] Holmes GP, Kaplan JE, Gantz NM, et al. Chronic Fatigue Syndrome: a working case definition. Ann Intern Med 1988; 108: 387-9. [3] Evengard B, Schacterle RS, Komaroff AL. Chronic Fatigue Syndrome: new insights and old ignorances. J Intern Med 1999; 246: 455-69. [4] McCully KK, Sisto SA, Natelson BH. Use of exercise for treatment of chronic fatigue syndrome. Sports Med 1996; 21: 35-48. [5] van Middendorp H, Geenen R, Kuis W, Heijnen CJ, Sinnema G. Psychological adjustment of adolescent girls with chronic fatigue syndrome. Pediatrics 2001; 107: 1-8. [6] De Becker P, Roeykens J, Reynders M, McGregor N, De Meirleir K. Exercise capacity in chronic fatigue syndrome. Arch Intern Med 2000; 160: 3270-7. [7] Fishler B, Dendale P, Michiels V, Cluydts R, Kaufman L, De Meirleir K. Physical fatigability and exercise capacity in chronic fatigue syndrome: association with disability, somatization and psychopathology. J Psychosom Res 1997; 42: 369-78. [8] Riley MS, O'Brien CJ, McCluskey DR, Bell NP, Nicholls DP. Aerobic work capacity in patients with chronic fatigue syndrome. Brit Med J 1990; 301: 953-6. [9] Sisto SA, LaManca J, Cordero DL, Bergen MT, Ellis SP, Drastal S, et al. Metabolic and cardiovascular effects of a progressive exercise test in patients with chronic fatigue syndrome. Am J Med 1996; 100: 634-40. [10] Fulcher KY, White PD. Strength and physiological response to exercise in patients with chronic fatigue syndrome. J Neurol Neurosurg Neuropsych 2000; 69: 302-7. [11] Wong R, Lopaschuk G, Zhu G, Walker D, Catellier D, Burton D, et al. Skeletal muscle metabolism in the chronic fatigue syndrome: in vivo assessment by 31 P nuclear magnetic resonance spectroscopy. Chest 1992; 102: 1716-22. [12] Sargent C, Scroop GC, Nemeth PM, Burnet RB, Buckley JD. Maximal oxygen uptake and lactate metabolism are normal in chronic fatigue syndrome. Med Sci Sports Exerc 2002; 34: 51-6. [13] Rowbottom D, Keast D, Pervan Z, Morton A. The physiological response to exercise in chronic fatigue syndrome. J Chronic Fatigue Syndr 1998; 4: 33- 49. [14] Kent-Braun JA, Sharma KR, Weiner MW, Massie B, Miller RG. Central basis of muscle fatigue in chronic fatigue syndrome. Neurology 1993; 43: 125-31. [15] Nijs J, De Meirleir K, Wolfs S, Duquet W. Disability evaluation in chronic fatigue syndrome: associations between exercise capacity and activity limitations/participation restrictions. Clin Rehabil 2004; 18: 109-18. [16] Sorensen B, Streib JE, Strand M, Make B, Giclas PC, Fleshner M, et al. Complement activation in a model of chronic fatigue syndrome. J Allergy Clin Immunol 2003; 112: 397-403. [17] Snell CR, Vanness JM, Strayer DR, Stevens SR. Physical performance and prediction of 2-5A Synthetase/RNase L antiviral pathway activity in patients with chronic fatigue syndrome. In Vivo 2002; 16: 107-10. [18] Suhadolnik RJ, Reichenbach NL, Hitzges P, et al. Upregulation of the 2-5A synthetase/Rnase L antiviral pathway associated with chronic fatigue syndrome. Clin Infect Dis 1994; 18: S96-S104. [19] Suhadolnik RJ, Peterson DL, Cheney PR, et al. Biochemical dysregulation of the 2-5A synthetase/Rnase L antiviral defense pathway in chronic fatigue syndrome. J Chronic Fatigue Syndr 1999; 5: 223-42. [20] De Meirleir K, Bisbal C, Campine I, et al. A 37 kDa 2-5A binding protein as a potential biochemical marker for Chronic Fatigue Syndrome. Am J Med 2000; 108: 99-105. [21] Suhadolnik RJ, Peterson DL, O'Brien K, et al. Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome. J Interferon Cytokine Res 1997; 17: 377-85. [22] Bisbal C, Martinand C, Silhol M, Lebleu B, Salehzada T. Cloning and characterization of a RNase L inhibitor. J Biol Chem 1995; 270: 13308-17. [23] Martinand C, Montavon C, Salehzada T, Silhol M, Lebleu B, Bisbal C. RNase L inhibitor is induced during human immunodeficiency virus type 1 infection and down regulates the 2-5A/Rnase L pathway in human T Cells. J Virol 1999; 73: 290-6. [24] Demettre E, Bastide L, D'Haese A, et al. Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients. J Biol Chem 2002; 277: 35746-51. [25] Bennett V, Otto E, Kunimoto M, et al. Diversity of ankyrins in the brain. Biochem Soc Trans 1991; 19: 1034-9. [26] Englebienne P, Herst CV, De Smet K, D'Haes A, De Meirleir K. Interactions between RNase L ankyrin-like domain and ABC transporters as a possible origin for pain, ion transport, CNS and immune disorders of Chronic Fatigue Immune Dysfunction Syndrome. J Chronic Fatigue Syndr 2001; 8: 83-102. [27] Nijs J, Demanet C, McGregor N, De Becker P, Verhas P, Englebienne P, et al. Monitoring a hypothetical channelopathy in chronic fatigue syndrome: preliminary observations. J Chronic Fatigue Syndr 2003; 11: 117-33. [28] Bisbal C, Silhol M, Laubenthal H, et al. The 2-5 oligoade-nylate/RNase L/ RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation. Mol Cell Biol 2000; 20: 4959-69. [29] Englebienne P. RNase L in health and disease - What did we learn recently? J Chronic Fatigue Syndr 2003; 11(2): 97-109. [30] De Becker P, McGregor N, De Meirleir K. A definition-based analysis of symptoms in a large cohort of patients with chronic fatigue syndrome. J Intern Med 2001; 250: 234-40. [31] Nasralla M, Haier J, Nicolson GL. Multiple mycoplasmal infections detected in blood of patients with chronic fatigue syndrome. Eur J Clin Microbiol Infect Dis 1999; 18: 859-65. [32] Vojdani A, Choppa PC, Tagle C, Andrin R, Samini B, Lapp CW. Detection of Mycoplasma genus and Mycoplasma fermentans by PCR in patients with Chronic fatigue Syndrome. FEMS Immunol Med Microbiol 1998; 22: 355-65. [33] Nijs J, Nicolson GL, De Becker P, Coomans D, De Meirleir K. High prevalence of Mycoplasma infections among European chronic fatigue syndrome patients. Examination of four Mycoplasma species in blood of chronic fatigue syndrome patients. FEMS Immunol Med Microbiol 2002; 34: 209-14. [34] Baseman J, Tully J. Mycoplasmas: sophisticated, reemerging, and burdened by their notority. Emerg Infect Dis 1997; 3: 21-32. [35] Baum SC. Mycoplasma pneumoniae and atypical pneumonia. In: Mandell GL, Bennet JE, Dolin R, editors. Principles and practice of infectious diseases. New York: Churchill Livingstone; 1995. p. 1713-8. [36] Peterszegi G, Texier S, Robert L. Cell death by overload of the elastin- laminin receptor on human activated lymphocytes: protection by lactose and melibiose. Eur J Clin Invest 1999; 29: 166-72. [37] Dabbagh K, Laurent GJ, Shock A, Leoni P, Papakrivopoulou J, Chambers RC. Alpha-1-antitrypsin stimulates fibroblast proliferation and procollagen production and activates classical MAP kinase signalling pathways. J Cell Physiol 2001; 186: 73-81. [38] Nijs J, De Meirleir K, Coomans D, De Becker P, Nicolson GL. Deregulation of the 2,5A synthetase RNase L antiviral pathway by Mycoplasmas in subsets of Chronic fatigue Syndrome. J Chronic Fatigue Syndr 2003; 11(2): 37-50. [39] Englebienne P, Fr emont M, Vaeyens F, Herst V, Verhas M, De Becker P, et al. Chronic fatigue syndrome (CFS) and multiple sclerosis (MS) as subsets of a group of cellular immunity disorders. ME/CFS: the medical practitioners' challenge. In: Proceedings at Sydney International Conference, 2001. p. 35-42. [40] De Meirleir K, De Becker P, Nijs J, Peterson D, Nicolson G, Patarca R, et al. Chronic Fatigue Syndrome etiology, the immune system and infection. In: Englebienne P, De Meirleir K, editors. Chronic Fatigue Syndrome: a biological approach. Boca Raton: CRC Medical Publications; 2002. p. 201-28 [chapter 8]. [41] Iordanov MS, Wong J, Bell JC, Magun BE. Activation of NF-kappaB by double- stranded RNA (dsRNA) in the absence of protein kinase R and RNase L demonstrates the existence of two separate dsRNA-triggered antiviral programs. Mol Cell Biol 2001; 21: 61-72. [42] Uetani K, Der SD, Zamanian-Daryoush M, de la Motte C, Lieberman BY, Williams BRG, Erzurum SC. Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase. J Immunol 2000; 165: 988-96. [43] Paludan SR, Ellermann-Eriksen S, Mogensen SC. NF-kappaB activation is responsible for the synergistic effect of herpes simplex virus type 2 infection on interferon-gamma-induced nitric oxide production in macrophages. J Gen Virol 1998; 79: 2785-93. [44] Vecchiet J, Cipollone F, Falasca K, Mezzetti A, Pizzigallo E, Bucciarelli T, et al. Relationship between musculoskeletal symptoms and blood markers of oxidative stress in patients with chronic fatigue syndrome. Neuroscience Letters 2003; 335: 151-4. [45] Kurup RK, Kurup PA. Hypothalamic digoxin, cerebral chemical dominance and myalgic encephalomyelitis. Intern J Neuroscience 2003; 113: 683-701. [46] Pall ML, Satterle JD. Elevated nitric oxide/peroxynitrite mechanism for the common etiology of multiple chemical sensitivity, chronic fatigue syndrome, and posttraumatic stress disorder. Ann NY Acad Sci 2001; 933: 323-9. [47] Casey AK, McDonough P, Finley MR, Behnke BJ, Richardson TE, Marlin DJ, et al. NO inhalation reduces pulmonary arterial pressure but not hemorrhage in maximal exercising horses. J Appl Physiol 2001; 91: 2674-8. [48] Silverthorn DU, Ober WC, Garrison CW, Silverthorn AC. Blood flow and the control of blood pressure. In: Silverthorn DU, Ober WC, Garrison CW, Silverthorn AC, editors. Human physiology: an integrated approach. New Jersey: Prentice Hall; 2001. p. 445-6 [chapter 15]. [49] Halliwill JR, Minson CT, Joyner MJ. Effect of systemic nitric oxide synthase inhibition on postexercise hypotension in humans. J Appl Phsyiol 2000; 89: 1830-6. [50] Scarim AL, Arnush M, Blair LA, Concepcion J, Heitmeier MR, Scheuner D, et al. Mechanisms of beta-cell death in response to double-stranded (ds) RNA and interferon-gamma. Am J Pathol 2001; 159: 273-83. [51] 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. Neuromusc Dis 2003; 13: 479-84. [52] Piec G, Mirkovitch J, Palacio S, et al. Effect of MALP-2, a lipopeptide from Mycoplasma fermentans, on bone resorption in vitro. Infect Immun 1999; 67: 6281-5. [53] Rawadi G. Mycoplasma fermentans interaction with mono-cytes/macrophages: molecular basis. Microbes Infect 2000; 2: 955-64. [54] Takeuchi O, Kaufmann A, Grote K, et al. Cutting edge: preferentially the R-stereoisomer of the Mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2-and MyD88-dependent signalling pathway. J Immunol 2000; 164: 554-7. [55] Rawadi G, Garcia J, Lemercier B, Roman-Roman S. Signal transduction pathways involved in the activation of NF-kappaB, AP-1, and c-fos by Mycoplasma fermentans membrane lipoproteins in macrophages. J Immunol 1999; 162: 2193-203. [56] Feng S-H, Tsai S, Rodriguez J, Lo S-C. Mycoplasmal infections prevent apoptosis and induce malignant transformation of interleukin-3-dependent 32D hematopoietic cells. Mol Cell Biol 1999; 19: 7995-8002. [57] Garcia J, Lemercier B, Roman-Roman S, Rawadi G. A Mycoplasma fermentans- derived synthetic lipopeptide induces AP-1 and NF-kappaB activity and cytokine secretion in macrophages via the activation of mitogen-activated protein kinase pathways. J Biol Chem 1998; 273: 34391-8. [58] Feng S-H, Lo S-C. Lipid extract of Mycoplasma penetrans proteinase K- digested lipid-associated membrane proteins rapidly activates NF-kappaB and activator protein 1. Infect Immun 1999; 67: 2951-6. [59] Kikkawa S, Matsumoto M, Sasaki T, Nishiguchi M, Tanaka K, Toyoshima K, et al. Complement activation in Mycoplasma fermentans-induced Mycoplasma clearance from infected cells: probing of the organism with monoclonal antibodies against M161Ag. Infect Immun 2000; 68: 1672-80. [60] Chia JKS, Chia LY. Chronic Chlamydia pneumoniae infection: a treatable cause of chronic fatigue syndrome. Clin Infect Dis 1999; 29: 452-3. [61] Nicolson GL, Gan R, Haier J. Multiple co-infections (Mycoplasma, Chlamydia, human herpes virus-6) in blood of chronic fatigue syndrome patients: association with signs and symptoms. APMIS 2003; 111: 557-66. [62] Levine S. Prevalence in the cerebrospinal fluid of the following infectious agents in a cohort of 12 CFS subjects: Human Herpes Virus-6 and 8; Chlamydia species; Mycoplasma species; EBV; CMV; and Coxsackievirus. J Chronic Fatigue Syndr 2001; 9: 41-51. [63] Fischer SF, Schwarz C, Vier J, H acker G. Characterization of antiapoptotic activities of Chlamydia pneumoniae in human cells. Infect Immun 2001; 69: 7121-9. [64] Dechend R, Maass M, Gieffers J, Dietz R, Scheidereit C, Leutz A, et al. Chlmaydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappaB and induces tissue factor and PAI-1 expression. Circulation 1999; 100: 1369-73. [65] Miller SA, Selzman CH, Shames BD, Barton HA, Johnson SM, Harken AH. Chlamydia pneumoniae activates nuclear factor jB and activator protein 1 in human vascular smooth muscle and induces cellular proliferation. J Surg Res 2000; 90: 76-81. [66] Molestina RE, Miller RD, Lentsch AB, Ramirez JA, Summersgill JT. Requirement for NF-kappaB in transcriptional activation of monocyte chemotactic protein 1 by Chlmay-dia pneumoniae in human endothelial cells. Infect Immun 2000; 68: 4282-8. [67] Wahl C, Oswald F, Simnacher U, Weiss S, Marre R, Essig A. Survival of Chlmaydia pneumoniae-infected mono mac 6 cells is dependent on NF-kappaB binding activity. Infect Immun 2001; 69: 7039-45. [68] Zaragoza C, Ocampo CJ, Saura M, McMillan A, Lowenstein CJ. Nitric oxide inhibition of coxsackievirus replication in vitro. J Clin Invest 1997; 100: 1760-7. [69] Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 1994; 79: 1137-46. [70] Bell EJ, McCartney RA, Riding MH. Coxsackie B viruses and myalgic encephalomyeltitis. J R Soc Med 1988; 81: 329-31. [71] Yousef GE, Bell EJ, Mann GF, Murugesan V, Smith DG, McCartney RA. Chronic enterovirus infection in patients with postviral fatigue syndrome. Lancet 1988; 1: 146-50. [72] Levy J. Viral studies of chronic fatigue syndrome, introduction. Clin Infect Dis 1994; 18(suppl 1): S117. -------- (c) 2004 Elsevier/Science Direct