Category: Biomarker

Utility of the blood for gene expression profiling and biomarker discovery in chronic fatigue syndrome

Abstract:

Chronic fatigue syndrome (CFS) is a debilitating illness lacking consistent anatomic lesions and eluding conventional laboratory diagnosis. Demonstration of the utility of the blood for gene expression profiling and biomarker discovery would have implications into the pathophysiology of CFS. The objective of this study was to determine if gene expression profiles of peripheral blood mononuclear cells (PMBCs) could distinguish between subjects with CFS and healthy controls.

Total RNA from PBMCs of five CFS cases and seventeen controls was labeled and hybridized to 1764 genes on filter arrays. Gene intensity values were analyzed by various classification algorithms and nonparametric statistical methods. The classification algorithms grouped the majority of the CFS cases together, and distinguished them from the healthy controls.

Eight genes were differentially expressed in both an age-matched case-control analysis and when comparing all CFS cases to all controls. Several of the differentially expressed genes are associated with immunologic functions (e.g., CMRF35 antigen, IL-8, HD protein) and implicate immune dysfunction in the pathophysiology of CFS. These results successfully demonstrate the utility of the blood for gene expression profiling to distinguish subjects with CFS from healthy controls and for identifying genes that could serve as CFS biomarkers.

 

Source: Vernon SD, Unger ER, Dimulescu IM, Rajeevan M, Reeves WC. Utility of the blood for gene expression profiling and biomarker discovery in chronic fatigue syndrome. Dis Markers. 2002;18(4):193-9. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3851413/ (Full article)

 

The head-up tilt test with haemodynamic instability score in diagnosing chronic fatigue syndrome

Abstract:

BACKGROUND: Studying patients with chronic fatigue syndrome (CFS), we have developed a method that uses a head-up tilt test (HUTT) to estimate BP and HR instability during tilt, expressed as a ‘haemodynamic instability score’ (HIS).

AIM: To assess HIS sensitivity and specificity in the diagnosis of CFS.

DESIGN:  Prospective controlled study.

METHODS: Patients with CFS (n=40), non-CFS chronic fatigue (n=73), fibromyalgia (n=41), neurally mediated syncope (n=58), generalized anxiety disorder (n=28), familial Mediterranean fever (n=50), arterial hypertension (n=28), and healthy subjects (n=59) were evaluated with a standardized head-up tilt test (HUTT). The HIS was calculated from blood pressure (BP) and heart rate (HR) changes during the HUTT.

RESULTS: The tilt was prematurely terminated in 22% of CFS patients when postural symptoms occurred and the HIS could not be calculated. In the remainder, the median(IQR) HIS values were: CFS +2.14(4.67), non-CFS fatigue -3.98(5.35), fibromyalgia -2.81(2.62), syncope -3.7(4.36), generalized anxiety disorder -0.21(6.05), healthy controls -2.66(3.14), FMF -5.09(6.41), hypertensives -5.35(2.74) (p<0.0001 vs. CFS in all groups, except for anxiety disorder, p=NS). The sensitivity for CFS at HIS >-0.98 cut-off was 90.3% and the overall specificity was 84.5%.

DISCUSSION: There is a particular dysautonomia in CFS that differs from dysautonomia in other disorders, characterized by HIS >-0.98. The HIS can reinforce the clinician’s diagnosis by providing objective criteria for the assessment of CFS, which until now, could only be subjectively inferred.

Comment in:

The head-up tilt test for diagnosing chronic fatigue syndrome. [QJM. 2003]

Assessing chronic fatigue. [QJM. 2003]

 

Source: Naschitz JE, Rosner I, Rozenbaum M, Naschitz S, Musafia-Priselac R, Shaviv N, Fields M, Isseroff H, Zuckerman E, Yeshurun D, Sabo E. The head-up tilt test with haemodynamic instability score in diagnosing chronic fatigue syndrome.  QJM. 2003 Feb;96(2):133-42. http://qjmed.oxfordjournals.org/content/96/2/133.long (Full article)

 

Hemodynamics instability score in chronic fatigue syndrome and in non-chronic fatigue syndrome

Erratum in: Semin Arthritis Rheum. 2003 Apr;32(5):343. Madelain, Fields [corrected to Fields, Madeline]; Hillel, Isseroff [corrected to Isseroff, Hillel].

 

Abstract:

OBJECTIVE: In studying patients with chronic fatigue syndrome (CFS) we developed a method that confers numerical expression to the degree of blood pressure and heart rate lability, ie, the ‘hemodynamic instability score’ (HIS). The HIS in CFS patients differed significantly from healthy subjects. The present investigation compares the HIS in CFS, non-CFS chronic fatigue and patients with recurrent syncope.

METHODS: Patients with CFS (n = 21), non-CFS chronic fatigue (n = 24), syncope of unknown cause (n = 44), and their age and sex-matched healthy controls (n = 21) were evaluated with a standardized head-up tilt test (HUTT). Abnormal reactions (endpoints) on HUTT were classified ‘clinical outcomes’ (cardioinhibitory or vasodepressor reaction, orthostatic hypotension, postural tachycardia syndrome) and ‘HIS endpoint’, i.e. HIS >-0.98.

RESULTS: The highest incidence of endpoints was noted in patients with CFS (79%), followed by patients with syncope of unknown cause (46%), non-CFS chronic fatigue (35%), and healthy subjects (14%). Presyncope or syncope during tilt occurred in 38% of CFS patients, 21% of patients with non-CFS chronic fatigue, and 43% of patients with recurrent syncope. The average HIS values were: CFS = +2.02 (SD 4.07), non-CFS chronic fatigue = -2.89 (SD 3.64), syncope = -3.2 (SD 3.0), healthy = -2.48 (4.07). The odds ratios for CFS patients to have HIS >-0.98 was 8.8 compared with non-CFS chronic fatigue patients, 14.6 compared with recurrent syncope patients, and 34.8 compared with healthy subjects.

CONCLUSION: The cardiovascular reactivity in patients with CFS has certain features in common with the reactivity in patients with recurrent syncope or non-CFS chronic fatigue, such as the frequent occurrence of vasodepressor reaction, cardioinhibitory reaction, and postural tachycardia syndrome. Apart from to these shared responses, the large majority of CFS patients exhibit a particular abnormality which is characterized by HIS values >-0.98. Thus, HIS >-0.98 lends objective criteria to the assessment of CFS.

Copyright 2002, Elsevier Science (USA). All rights reserved.

 

Source: Naschitz JE, Sabo E, Naschitz S, Rosner I, Rozenbaum M, Fields M, Isseroff H, Priselac RM, Gaitini L, Eldar S, Zukerman E, Yeshurun D. Hemodynamics instability score in chronic fatigue syndrome and in non-chronic fatigue syndrome. Semin Arthritis Rheum. 2002 Dec;32(3):141-8. http://www.ncbi.nlm.nih.gov/pubmed/12528078

 

IgM serum antibodies to human cytomegalovirus nonstructural gene products p52 and CM2(UL44 and UL57) are uniquely present in a subset of patients with chronic fatigue syndrome

Abstract:

Human cytomegalovirus (HCMV) IgM serum antibodies to two nonstructural gene products UL44 and UL57 (p52 and CM2) were assayed in patients with the diagnosis of the chronic fatigue syndrome (CFS) according to criteria established by the US Centers for Disease Control and Prevention. A subset of 16 CFS patients demonstrated HCMV IgG, but no HCMV IgM serum antibodies to conformational structural HCMV antigens (designated, V). By convention, these findings are interpreted to indicate only a remote HCMV infection.

However, HCMV IgM p52 and CM2 antibodies were uniquely present in these 16 CFS patients. Other CFS patients with similar HCMV (V) IgG antibodies (18 patients), non-fatigued HCMV (V) IgG-positive control patients (18 patients), random HCMV (V) IgG-positive control patients from a clinical laboratory (26 patients), and non-fatigued HCMV (V) IgG-negative control patients (15 patients) did not have HCMV, IgM p52 or CM2 serum antibodies (p < 0.05). Control HCMV (V) IgG-positive patients had no serum IgM HCMV (V) antibodies to conventional structural HCMV (V) antigen. Thus, 77 various control patients did not contain IgM p52 or CM2 serum antibodies. The presence of IgM p52 and/or CM2 HCMV serum antibodies in this subset of CSF-specific patients may detect incomplete HCMV multiplication in which a part of the HCMV protein-coding content of the HCMV genome is processed, but remains unassembled.

These findings suggest that the presence of HCMV IgM p52 and CM2 serum antibodies may be a specific diagnostic test for the diagnosis of a subset of CFS patients. Further, these data suggest an etiologic relationship for HCMV infection in this group of CFS patients

 

Source: Lerner AM, Beqaj SH, Deeter RG, Fitzgerald JT. IgM serum antibodies to human cytomegalovirus nonstructural gene products p52 and CM2(UL44 and UL57) are uniquely present in a subset of patients with chronic fatigue syndrome. In Vivo. 2002 May-Jun;16(3):153-9. http://www.ncbi.nlm.nih.gov/pubmed/12182109

 

Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients

Abstract:

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.

 

Source: Demettre E, Bastide L, D’Haese A, De Smet K, De Meirleir K, Tiev KP, Englebienne P, Lebleu B.Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients. J Biol Chem. 2002 Sep 20;277(38):35746-51. Epub 2002 Jul 12. http://www.ncbi.nlm.nih.gov/pubmed/12118002

 

Physical performance and prediction of 2-5A synthetase/RNase L antiviral pathway activity in patients with chronic fatigue syndrome

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.

 

Source: 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 Mar-Apr;16(2):107-9. http://www.ncbi.nlm.nih.gov/pubmed/12073768

 

Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome

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.

 

Source: Shetzline SE, Martinand-Mari C, Reichenbach NL, Buletic Z, Lebleu B, Pfleiderer W, Charubala R, De Meirleir K, De Becker P, Peterson DL, Herst CV,Englebienne P, Suhadolnik RJ. Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome.  J Interferon Cytokine Res. 2002 Apr;22(4):443-56. http://www.ncbi.nlm.nih.gov/pubmed/12034027

 

Antiviral pathway activation in chronic fatigue syndrome and acute infection

Comment on: Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection. [Clin Infect Dis. 2001]

 

SIR—We read the very engaging report by Gow et al. [1] with the utmost interest. However, we feel that this article raises more questions than clear-cut answers regarding the hypothesis that motivated the study—that is, that the previously reported activation of the antiviral pathway in chronic fatigue syndrome (CFS) might be linked to infection rather than to CFS specifically. To verify their hypothesis, Gow and colleagues used PCR to measure the genetic expression of 3 IFN-regulated genes—namely, the latent ribonuclease (RNase L), RNA-regulated protein kinase (PKR), 2,5 synthetase, and the RNase L inhibitor (RLI)—in patients with acute infection (in their study, severe gastroenteritis; group 1), patients with CFS (group 2), and healthy control subjects (group 3).

First, surprisingly enough, although they recognized that acute infection is supposed to induce the expression of the genes selected for their study (see figure 1 of [1]), Gow and colleagues failed to find any significant increase in the expression of 2 major genes (RNase L and 2,5 synthetase) in group 1, as compared with groups 2 and 3; they observed only increased mRNA for PKR and RLI. Although it is recognized that genetic expression of PKR, RNase L, and 2,5 synthetase is under the control of interferon, RLI is definitely not [2]. Upregulation of RLI genetic expression with a normal genetic expression of both 2,5 synthetase and RNase L (although PKR is overexpressed!) during acute infection, as was observed in the study of Gow et al. [1], would indicate not only that RNase L is not activated (normal expression of RNase L and, more importantly, of 2,5 synthetase), but that it is further inhibited by an overexpressed RLI [2]. Such a scenario, if verified, would be in complete disagreement with the current understanding of the IFN pathway [3]. Therefore, we cannot help but wonder how Gow and colleagues reconcile their observations with the acute infection status of study group 1. In our view, this inconsistency severely undermines their conclusions.

You can read the rest of this comment here:  http://cid.oxfordjournals.org/content/34/10/1420.long

 

Source: De Meirleir K, Suhadolnik RJ, Lebleu B, Englebienne P. Antiviral pathway activation in chronic fatigue syndrome and acute infection. Clin Infect Dis. 2002 May 15;34(10):1420-1; author reply 1421-2. http://cid.oxfordjournals.org/content/34/10/1420.long (Full article)

 

Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection

Abstract:

Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS.

Comment in: Antiviral pathway activation in chronic fatigue syndrome and acute infection. [Clin Infect Dis. 2002]

 

Source: Gow JW, Simpson K, Behan PO, Chaudhuri A, McKay IC, Behan WM. Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection. Clin Infect Dis. 2001 Dec 15;33(12):2080-1. Epub 2001 Nov 6. http://cid.oxfordjournals.org/content/33/12/2080.long (Full article)

 

Characterization of a 2′,5′-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation

Erratum in: J Biol Chem 2001 Aug 24;276(34):32392.

Abstract:

Upregulation of key components of the 2′,5′-oligoadenylate (2-5A) synthetase/RNase L pathway has been identified in extracts of peripheral blood mononuclear cells from individuals with chronic fatigue [corrected] syndrome, including the presence of a low molecular weight form of RNase L. In this study, analysis of 2′,5′-Oligoadenylate (2-5A) binding and activation of the 80- and 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80- and the 37-kDa RNase L with the 2-5A azido photoprobe, [(32)P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [(32)P]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 (p(3)A(3)) resulted in complete protection against photolabeling, demonstrating that [(32)P]pApAp(8-azidoA) binds specifically to the 2-5A-binding site of the 80- and 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 [(32)P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.

 

Source: Shetzline SE, Suhadolnik RJ. Characterization of a 2′,5′-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation. J Biol Chem. 2001 Jun 29;276(26):23707-11. Epub 2001 Apr 25. http://www.jbc.org/content/276/26/23707.long (Full article)