Abstract: The elevated RNase L enzyme activity observed in some Chronic Fatigue Syndrome (CFS) patients may be linked to the low exercise tolerance and functional impairment that typify this disease. The purpose of this investigation was to determine if specific indicators of physical performance can predict abnormal RNase L activity in CFS patients. Seventy-three CFS patients performed a graded exercise test to voluntary exhaustion. Forty-six patients had elevated RNase L levels. This measure was employed as the dependent variable in a discriminant function analysis, with peak V02, exercise duration and Karnofsky Performance Scores (KPS) serving as the independent variables. All three variables entered the single significant function (p < 0.001). The elevated RNase L group had a lower peak V02 and duration than the normal group, but a higher KPS. The results suggest that both exercise testing and the RNase L biomarker have potential to aid in the diagnosis of CFS.

RNase L in Health and Disease -- What Did We Learn Recently? Patrick Englebienne J of Chronic Fatigue Syndrome, Vol. 11(2) 2003, pp. 97-109 Patrick Englebienne is affiliated with the Department of Nuclear Medicine, Free University of Brussels, Brugmann University Hospital, Place van Gehuchten 4, B-1O20 Brussels, Belgium, and RED Laboratories N. V., Pontbeek 61, B-1731 Zellik, Belgium (E-mail: mailto:penglebi@ulb.ac.be ). RECEIVED: 09/02/02 REVISED: 09/12/02 ACCEPTED: 09/16/02

ABSTRACT. The 2',5'-oligoadenylate-dependent ribonuclease L (RNase L) is central to the innate cellular defense mechanism induced by type I interferons during intracellular infection. The protein, activated by 2',5'-oligoadenylates precludes the replication of the infectious agent by cleaving single-stranded RNA and, along with the double-stranded RNA-dependent protein kinase, its spreading by inducing the cell to undergo suicide (apoptosis). In absence of infection, the protein remains dormant. Recent evidence indicates, however, that the protein is activated in absence of infection and may play a role in cell differentiation, immune activation, and act as a tumor-suppressor. A deregulation in this pathway has been documented in immune cells of chronic fatigue syndrome patients which involves the presence of a catalytically active truncated RNase L. This protein escapes the normal regulation which implies the development of a cascade of unwanted cellular events. The present article reviews our current understanding of this deregulation, enlightens its relevance in the pathological process and proposes new targets for therapeutic development.

Biochemical Dysregulation of the 2-5A Synthetase/RNase L Antiviral Defense Pathway in Chronic Fatigue Syndrome Robert J. Suhadolnik, PhD; Daniel L. Peterson, MD; Paul R. Cheney, MD, PhD; Susan E. Horvath, BS; Nancy L. Reichenbach, BS; Karen O'Brien, BS; Vincent Lombardi, BA; Suzanne Welsch, MS; Elizabeth G. Furr, BS; Ramamurthy Charubala, PhD; Wolfgang Pfleiderer, PhD [ Journal of Chronic Fatigue Syndrome (The Haworth Medical Press, an imprint of The Haworth Press, Inc.) Vol. 5, No. 3/4, 1999 ]

SUMMARY. The aim of the current study was to examine the biochemical defects in key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L antiviral pathway in an extended cohort of patients with chronic fatigue syndrome (CFS) from two sites. CFS patients, who met the CDC criteria for CFS, and matched controls were assessed with respect to their general health, depression, and pain. Biochemical assays were completed for three blood draws over a period of one year. Analysis of the mean values for bioactive 2-5A, RNase L activity, low molecular weight (LMW) RNase L in CFS PBMC extracts confirmed the statistically significant upregulation of the 2-5A synthetase/RNase L pathway compared to control PBMC extracts (p = .001, .002, and .007, respectively). Clinical correlates to the biochemical findings included a negative correlation between Karnofsky Performance Score and bioactive 2-5A (p = .025) or RNase L activity (p = .002) and positive correlation between Metabolic Screening Questionnaire and RNase L activity (p = .01) and between interferon- and LMW RNase L (p = .05). The evidence presented in this study more firmly establishes the dysregulation of the 2-5A synthetase/RNase L pathway in CFS.

Characterization of a 2-5A dependent 37-kDa RNase L. 2. Azido photoaffinity labeling and 2-5A Dependent activation. Shetzline SE, Suhadolnik RJ. J Biol Chem 2001 Apr 25; [epub ahead of print] Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140. PMID: 11323422

The preceding paper in this issue described the characterization of the molecular structure of the 37-kDa RNase L identified in peripheral blood mononuclear cell (PBMC) extracts from individuals with chronic fatigue syndrome (CFS) [Shetzline, S., et al., (2001) J. Biol. Chem. (preceding paper in this issue)].

In this study, analysis of 2-5A binding and activation of the 80-kDa and the 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80-kDa and the 37-kDa RNase L with the 2-5A azido photoprobe, [32P]pApAp(8-azidoA), was achieved.

Half-maximal photoinsertion of [32P]pApAp(8-azidoA) occurred at 3.7 x 10-8 M for the 80-kDa RNase L and at 6.3 x 10-8 M for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2-5A photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p3A3) resulted in complete protection against photolabeling, demonstrating that [32P]pApAp(8-azidoA) binds specifically to the 2-5A binding site of the 80-kDa and the 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa RNase L was three times faster than the 80-kDa RNase L.

The data obtained from these 2-5A binding and 2-5A-dependent activation studies demonstrate the utility of [32P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in PBMC extracts.

G-Actin Cleavage Parallels 2-5-A-Dependent RNase L Cleavage in Peripheral Blood Mononuclear Cells– Relevance to a Possible Serum-Based Screening Test for Dysregulations in the 2-5A Pathway Simon Roelens, C Vincent Herst, Anne D'Haese, Karen De Smet, Marc Fremont, Kenny De Meirleir, Patrick Englebienne Innov in Chronic Fatigue Syndrome Res and Clin Practice 2001; 8(3-4):63-82.

Summary: A dysregulation in the 2',5'-oligoandenylate (2-5A)-dependent RNase L antiviral pathway has been detected in peripheral blood mononuclear cells (PBMC) of chronic fatigue syndrome (CFS) patients, which is characterized by an unregulated RNase L activity and the presence of a low molecular weight (LMW) 2-5A-binding protein (37-kDa 2-5A-BP). This study was undertaken to test the possibility that the 37-kDa 2-5A-BP Of CFS is produced by proteolytic cleavage of the 80-kDa monomeric enzyme. Incubation of the 80-kDa human recombinant RNase L (r-hRNase L) with PBMC extracts either positive or negative for the presence of 37-kDa 2-5A-BP, respectively, demonstrates that the LMW protein is produced by the former, not the latter, and that the size of the fragment generated from the recombinant protein matches the 37-kDa size of the fragment observed in the original PBMC. Digestion of r-hRNase L with calpain generated the same 37-kDa 2-5A-BP observed in PBMC extracts and calpain immunoprecipitation from PBMC extracts reduced their proteolytic activity, an observation that suggests that calpain may be involved in the cleavage. We further examined G-actin, a known calpain substrate, for possible cleavage in PBMC. Actin fragments were observed of which the presence correlated with the presence of 37-kDa 2-5A-BP. Since G-actin is cleared by serum transport, we further screened serum samples for the presence of LMW forms. A single LMW actin fragment could be detected in serum, the presence of which correlated significantly with the presence of both G-actin and RNase L fragments in PBMC. This latter observation offers the opportunity to screen large populations of patients for dysregulations in the RNase L pathway by a serum-based assay.

Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome. Susan Shetzline, Camille Martinand-Mari, Nancy L Reichenbach, Zivjena Buletic, Bernard Lebleu, Wolfgang Pfleiderer, Ramamurthy Charubala, Kenney De Meirleir, Pascale De Becker, Daniel L Peterson, CVT Herst, Patrick Englebienne, Robert J Suhadolnik. Department of Biochemistry and the Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA. J Interferon and Cytokine Research 2002; 22(4):443-456

Abstract: A 2',5'-oligoadenylate (2-5A)-dependent 37-kDa form of RNase L has been reported in extracts of peripheral blood mononuclear cells (PBMC) from individuals with chronic fatigue syndrome (CFS). In the current study, analytic gel permeation FPLC, azido photoaffinity labeling, two-dimensional (2-D) gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been used to examine the biochemical relationship between the 80-kDa RNase L in healthy control PBMC and the 37-kDa RNase L in PBMC from individuals with CFS. Like the 80-kDa RNase L, the 37-kDa RNase L is present as a catalytically inactive heterodimer complex with the RNase L inhibitor (RLI). Formation of a 37-kDa RNase L-RLI complex indicates that the 37-kDa RNase L is structurally similar to the 80-kDa RNase L at the N-terminus, which contains the 2-5A binding domain. The enzymatically active monomer form of 37-kDa RNase L resolved by 2-D gel electrophoresis has a pI of 6.1. RT-PCR and Southern blot analyses demonstrated that the 37-kDa RNase L is not formed by alternative splicing. In-gel tryptic digestion of the 37-kDa RNase L that was excised from 2-D gels and subsequent MALDI-MS analysis identified three peptide masses that are identical to three predicted peptide masses in the 80-kDa RNase L. The electrophoretic mobility of 2-5A azido photolabeled/immunoprecipitated 37-kDa RNase L was the same under reducing and nonreducing conditions. The results presented show that the 37-kDa form of RNase L in PBMC shares structural and functional features with the native 80-kDa RNase L, in particular in the 2-5A binding and catalytic domains.

Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome. Robert J Suhadolnik, Daniel L Peterson, K O'Brien, PR Cheney, CV Herst, Nancy L Reichenbach, N Kon, SE Horvath, KT Iacono, ME Adelson, Kenny De Meirleir, Pascale De Becker, Ramamurthy Charubala, Wolfgang Pfleiderer. Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA. J Interferon and Cytokine Research 1997; 17(7):377-385.

Abstract: Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.

Ribonuclease L Proteolysis in Peripheral Blood Mononuclear Cells of Chronic Fatigue Syndrome Patients Edith Demettre§, Lionel Bastide§¶, Anne D'Haese§¶, Karen De Smet¶, Kenny De Meirleir, Kiet P. Tiev**, Patrick Englebienne¶, and Bernard Lebleu§§ From the §UMR 5124 CNRS, Université Montpellier 2, 34293 Montpellier, France, ¶ R.E.D Laboratories, 1731 Zellik, Belgium, Department of Human Physiology and Medicine, Vrije Universiteit Brussel, 1090 Brussels, Belgium, ** Service de Médecine Interne, Hôpital Saint Antoine, 75571 Paris, France, and Department of Nuclear Medicine, Université Libre de Bruxelles, 1050 Brussels, Belgium J. Biol. Chem., Vol. 277, Issue 38, 35746-35751, September 20, 2002

A 37-kDa binding polypeptide accumulates in peripheral blood mononuclear cell (PBMC) extracts from chronic fatigue syndrome (CFS) patients and is being considered as a potential diagnostic marker (De Meirleir, K., Bisbal, C., Campine, I., De Becker, P., Salehzada, T., Demettre, E., and Lebleu, B. (2000) Am. J. Med. 108, 99-105).

We establish here that this low molecular weight 2-5A-binding polypeptide is a truncated form of the native 2-5A-dependent ribonuclease L (RNase L), generated by an increased proteolytic activity in CFS PBMC extracts.

RNase L proteolysis in CFS PBMC extracts can be mimicked in a model system in which recombinant RNase L is treated with human leukocyte elastase.

RNase L proteolysis leads to the accumulation of two major fragments with molecular masses of 37 and 30 kDa. The 37-kDa fragment includes the 2-5A binding site and the N-terminal end of native RNase L. The 30-kDa fragment includes the catalytic site in the C-terminal part of RNase L.

Interestingly, RNase L remains active and 2-5A-dependent when degraded into its 30- and 37-kDa fragments by proteases of CFS PBMC extract or by purified human leukocyte elastase.The 2-5A-dependent nuclease activity of the truncated RNase L could result from the association of these digestion products, as suggested in pull down experiments.

RNase L dysfunction disorder (R.E.D.D.) in CFS K. De Meirleir*, LCLI, I. Campine+*, P. De Becker, B. Van Steenberge*, C. Bisbal**, T. Salehzada**, B. Lebleu**, C.V. Herst**Department of Human Physiology, Free University of Brussels, Brussels, Belgium **Institute of Molecular Genetics, Montpellier University, Montpellier, France + I.Campine is supported by funds from the Foundation for Scientific Research, Belgium (F.W.O.).

Objective: The unknown aetiology and absence of biochemical markers in CFS are a major problem in this disorder. Activation of immune responses and infection by several viruses have been suggested in several studies. Recent work by Suhadolnik et al. has demonstrated increased levels of 2-5' A oligonucleotides, 2'-5'A Synthetase and RNase L activity in mononuclear cell pellets from CFS patients, as well as a low molecular form of RNase L in severely disabled CFS patients. This work was designed to explore the specificity and sensitivity of the presence of the LMW RNase L in CFS. Methods: Mononuclear cell pellets (PBMC) of 57 patients and 18 controls were studied. The technique used to detect the RNase L molecular weight is described by Charachon et al (Biochemistry 29:2550-2556, 1990). This technique is different from the one described by the one used by Suhadolnik et al. (Clin Inf Dis, 18 (Suppl 1), 5 96-104, 1994). An RLI binding study was also performed. Results: A low molecular weight (LMW) 2'-5'A binding polypeptide (37 kDa) was found in 50 out of 57 PBMC pellets of the CFS patients, versus 4 out of 18 healthy individuals. Both sensitivity and specificity of the LMW RNase L in relationship to CFS are high. The 37kDa 2'-5'A binding polypeptide binds RLI. Conclusion: The presence of a 37 kDa 2'.-5'A binding polypeptide in the PBMC pellets of CFS patients may objectively contribute to distinguish CFS patients from healthy individuals. These observations could provide the basis for the development of a biochemical assay for the differential diagnosis of CFS and for follow up of its clinical evolution.

A controlled trial with a specially configured RNA drug, Poly(I)åPoly(C12U), in chronic fatigue syndrome. Strayer DR, Carter WA, Brodsky I, Cheney P, Peterson D, Salvato P, Thompson C, Loveless M, Shapiro DE, Elsasser W, Gillespie DH. Clinical Infectious Diseases 1994; 18(Supp 1): S88-95.

Abstract: Chronic fatigue syndrome (CFS) is a physically debilitating illness associated with immunologic abnormalities, viral reactivation, and impairment of cognition. In a randomized, multicenter, placebo-controlled, double-blind study of 92 patients meeting the CFS case definition of the Centers for Disease Control and Prevention, the response of several laboratory and clinical variables to an antiviral and immunomodulatory drug, poly(I)åpoly(C12U), was determined. Measures of clinical response included Karnofsky performance score, a cognition scale derived from a self-administered instrument assessing symptomatology (SCL-90-R), an activities of daily living scale, and exercise treadmill performance. After 24 weeks, patients receiving poly(I)åpoly(C12U) had higher scores for both global performance and perceived cognition than did patients receiving placebo. In particular, patients given poly(I)åpoly(C12U) had increased Karnofsky performance scores (P < .03), exhibited a greater ability to do work during exercise treadmill testing (P=".01)," displayed an enhanced capacity to perform the activities of daily living (P < .04), had a reduced cognitive deficit (P=".05)," and required less use of other medications (P < .05).

Long term improvements in patients with chronic fatigue syndrome treated with Ampligen. Strayer DR, Carter W, Strauss KI, Brodsky I, Suhadolnik RJ, Ablashi D, Henry B, Mitchell WM, Bastein S, Peterson D. Journal of Chronic Fatigue Syndrome 1995; 1(1): 35-53.

Abstract: Fifteen patients who fit the CDC definition of chronic fatgiue syndrome (CFS) and had evidence of severe reduction in performance levels by low Karnofsky performance scores (KPS) of 20 - 60 were treated with Ampligen. At baseline most patients showed evidence of cerebral dysfunction by neuropsychological testing, were antigen positive by cell culture assay for human herpesvirus-6 (HHV-6), and displayed reduced performance during exercise tolerance testing, as measured by oxygen consumption. These patients represented a subset of CFS patients with especially severe and sustained symptomatology. Following 12 - 48 weeks of Ampligen therapy, sustained improvements were noted in KPS (p< .01) Cognitive function improved including IQ and memory. Oxygen uptake and treadmill duration during exercise tolerance testing was also improved after 24 weeks of treatment (p < .01). Reduction in HHV-6 expression as measured by the giant cell assay was significant (p < 0.001). Patients continued to show significant improvement late in therapy, taking 8 to 12 weeks as baseline. It was concluded that while receiving Ampligen, the severely afflicted patients studied here derived long-lasting clinical benefit from the Ampligen therapy.

Biochemical evidence for a novel low molecular weight 2-5A-dependent RNaseL in chronic fatigue syndrome. Suhadolnik RJ, Peterson DL, O'Brien K, Cheney PR, Herst CV, Reichenbach NL, Kon N, Horvath SE, Iacono KT, Adelson ME, De Meirleir K, De Becker P, Charubala R, Pfleiderer W. 1997; 17(7): 377-385.

Abstract: Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNaseL and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNaseL activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNaseL and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNaseL enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNaseL enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNaseL enzyme activity only at 30 kDa. Evidence is provided indicating that the RNaseL enzyme dysfunction in CFS is more complex than previously reported.

Changes in the 2-5A synthetase/RNaseL antiviral pathway in a controlled clinical trial with poly(I)-poly(C12U) [Ampligen] in CFS. Suhadolnik RJ, Reichenbach NL, Hitzges P, Adelson ME, Peterson DL, Cheney P, Salvato P, Thompson C, Loveless M, Muller WE, et al. In Vivo 1994; 8(4): 599-604.

Abstract: Latent 2', 5'-oligoadenylate (2-5A) synthetase activity, bioactive 2-5A and RNaseL activity were measured in extracts of peripheral blood mononuclear cells (PMBC) before and during a randomized, multicenter, placebo-controlled, double-blind study of poly(I)-poly(C12U) in individuals with chronic fatigue syndrome (CFS) as defined by the Centers for Disease Control and Prevention. The mean values for bioactive 2-5A and RNaseL activity were significantly elevated at baseline compared to controls (p < .0001 and p=".001," respectively). In individuals that presented with elevated RNaseL activity at baseline, therapy with poly(I)-poly(C12U) resulted in a significant decrease in both bioactive 2-5A and RNaseL activity (p=".09" and p=".005," respectively). Decrease in RNaseL activity in individuals treated with poly(I)-poly(C12U) correlated with cognitive improvement (p=".007)." Poly(I)-poly(C12U) therapy resulted in a significant decrease in bioactive 2-5A and RNaseL activity in agreement with clinical and neuropsychological improvements (Strayer DR, et al., Clin. Infectious Dis. 18:588-595, 1994). The results described show that poly(I)-poly(C12U) is a biologically active drug in CFS.