Category: Biomarker

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)

 

A 37 kDa 2-5A binding protein as a potential biochemical marker for chronic fatigue syndrome

Abstract:

PURPOSE: Recent studies have revealed abnormalities in the ribonuclease L pathway in peripheral blood mononuclear cells of patients with the chronic fatigue syndrome. We conducted a blinded study to detect possible differences in the distribution of 2-5A binding proteins in the cells of patients with chronic fatigue syndrome and controls.

PATIENTS AND METHODS: We studied 57 patients with chronic fatigue syndrome and 53 control subjects (28 healthy subjects and 25 patients with depression or fibromyalgia). A radioactive probe was used to label 2-5A binding proteins in unfractionated peripheral blood mononuclear cell extracts and to compare their distribution in the three groups.

RESULTS: A 37 kDa 2-5A binding polypeptide was found in 50 (88%) of the 57 patients with chronic fatigue syndrome compared with 15 (28%) of the 53 controls (P < 0.01). When present, the amount of 37 kDa protein was very low in the control groups. When expressed as the ratio of the 37 kDa protein to the 80 kDa protein, 41 (72%) of the 57 patients with chronic fatigue syndrome had a ratio > 0.05, compared with 3 (11%) of the 28 healthy subjects and none of the patients with fibromyalgia or depression.

CONCLUSION: The presence of a 37 kDa 2-5A binding protein in extracts of peripheral blood mononuclear cells may distinguish patients with chronic fatigue syndrome from healthy subjects and those suffering from other diseases.

Comment in:

The biology of chronic fatigue syndrome. [Am J Med. 2000]

Chronic fatigue syndrome: the fundamentals still apply. [Am J Med. 2000]

Is there a Gulf War syndrome? [Am J Med. 2000]

Chronic fatigue syndrome. [Am J Med. 2000]

 

Source: De Meirleir K, Bisbal C, Campine I, De Becker P, Salehzada T, Demettre E, Lebleu B. A 37 kDa 2-5A binding protein as a potential biochemical marker for chronic fatigue syndrome. Am J Med. 2000 Feb;108(2):99-105. http://www.ncbi.nlm.nih.gov/pubmed/11126321

 

Interferon-induced proteins are elevated in blood samples of patients with chemically or virally induced chronic fatigue syndrome

Abstract:

Overlapping symptomatologies between Chronic Fatigue Syndrome (CFS) and Chemical Sensitivity have been observed by different investigators. Therefore, it is of great importance to develop biomarker(s) for possible differentiation between viral induced CFS (without sensitivity to chemicals) versus chemically induced CFS.

Since interferon induced proteins 2-5A Synthetase and Protein Kinase RNA (PKR) have been implicated in the viral induction of CFS, the objective of this study was to utilize 2-5A and PKR activity for differentiation between CFS induced by either viruses or chemicals.

Based on the CDC definition and criteria, twenty CFS patients who were positive for viral genome(s) (mainly HHV6; HTLVII, EBV, and CMV) and did not have any history of exposure to toxic chemicals were included in this study. As a comparison, the second group of patients consisted of twenty individuals from the same geographical area who were negative for viral genomes but had been exposed to methyl tertiary-butyl ether concentration of up to 70 ppb and benzene concentration up to 14 ppb. All patients complained of fatigue and other symptoms overlapping between the two groups. From all 40 patients, blood was drawn, leukocyte extract was prepared and assayed for 2-5A Synthetase and PKR activity.

Clinical specimens which were positive for viral genomes showed from 2.2-38.7 fold increase in 2-5A activity and 1.3-13.5 fold increase in PKR activities over the background of the healthy controls. Similarly, the second group (negative for viral genomes, but exposed to chemicals) showed a 1.1-29.2 fold increase for 2-5A Synthetase and a 1.3-11.6 fold increase for PKR when they were compared to healthy subjects.

To elucidate mechanisms involved in viral versus chemical induction of 2-5A Synthetase and PKR, MDBK cell lines were cultured either in the presence or absence of HHV6, MTBE, or Benzene, heat shock proteins and interferon-beta. 2-5A and PKR activities were measured in all the above conditions.

A clear induction of 2-5A and PKR was observed when MDBK cells were exposed to HHV6, MTBE, and Benzene. This induction was more significant with HSP90, HSP70, and IFN-beta indicating their involvement in the mechanism of action. However, when MDBK cells were incubated either with MTBE + Benzene or HHV6 in the presence or absence of anti IFN-beta or anti-HSP-70, the activities of both 2-5A and PKR in HHV6 infected cells were inhibited by more than 90% due to addition of anti IFN-beta, and only 20% by addition of anti-HSP70. While in MTBE + Benzene exposed cells anti IFN-beta reduced the activity of these enzymes by 40% and anti-HSP70 by more than 90%.

This variation in the induction of 2-5A and PKR by anti-HSP70 or IFN-beta indicates involvement of IFN-beta in viral induction 2-5A and PKR, and HSP involvement in chemical induction of these enzymes. We conclude that 2-5A and PKR are not only biomarkers for viral induction of CFS, but biomarkers to other stressors that include MTBE and Benzene.

 

Source: Vojdani A, Lapp CW. Interferon-induced proteins are elevated in blood samples of patients with chemically or virally induced chronic fatigue syndrome. Immunopharmacol Immunotoxicol. 1999 May;21(2):175-202. http://www.ncbi.nlm.nih.gov/pubmed/10319275

 

Downregulation of RNase L inhibitor correlates with upregulation of interferon-induced proteins (2-5A synthetase and RNase L) in patients with chronic fatigue immune dysfunction syndrome

Abstract:

Chronic Fatigue Immune Dysfunction Syndrome (CFIDS) is a disorder characterized by debilitating fatigue associated with immunological abnormalities and cognitive impairments. The recently cloned RNase L Inhibitor (RLI) gene encodes a specific protein which is believed to regulate 2-5A synthetase and RNase L activity via the formation of a latent heterodimeric protein complex.

In the present study, we investigated the levels of 2-5A synthetase, RNase L and RLI in patients with CFIDS as compared to healthy controls. Quantitative Competitive PCR (Q/C PCR) analysis showed a statistically significant decrease in RLI mRNA present in the peripheral blood lymphocytes (PBL) of patients with CFIDS (n = 25, mean = 569, S.E = 154) as compared to RLI mRNA level present in peripheral blood lymphocytes (PBL) of healthy controls (n = 15, mean = 2296, S.E = 506; p < 0.0001).

The decrease in RLI mRNA in CFIDS individuals correlated directly with RLI and RLI: RNase L protein ratio while showing an inverse relationship to the 2-5A synthetase and RNase L activity. This RLI mRNA and protein deficiency in CFIDS patients may explain the increase in activity of RNase L found in CFIDS patients.

The unidirectional decrease in RLI message and protein levels in CFIDS individuals may contribute to the destabilization of the latent RLI:RNase L heterodimeric protein complex, resulting in the excessive activation of RNase L shown in this study.

The increased activation of RNase L may result in an increased cellular RNA turnover and subsequent inhibition of protein synthesis; thus resulting in general fatigue, myalgia muscle weakness and other symptomatologies shown in CFIDS patients.

Furthermore, this data supports the hypothesis that the antiviral 2-5 oligoadenylate synthetase (2-5OAS) overexpression in individuals with CFIDS correlates with an increase in RNase L activity and with a decrease in RNase L inhibitor.

 

Source: Vojdani A, Choppa PC, Lapp CW. Downregulation of RNase L inhibitor correlates with upregulation of interferon-induced proteins (2-5A synthetase and RNase L) in patients with chronic fatigue immune dysfunction syndrome. J Clin Lab Immunol. 1998;50(1):1-16. http://www.ncbi.nlm.nih.gov/pubmed/10189612