Tag: Lapp

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

 

Detection of Mycoplasma genus and Mycoplasma fermentans by PCR in patients with Chronic Fatigue Syndrome

Abstract:

Mycoplasma fermentans and other Mycoplasma species are colonizers of human mucosal surfaces and may be associated with human immunodeficiency virus infection. While many infectious agents have been described in different percentages of patients with Chronic Fatigue Syndrome (CFS), little is known about the prevalence of mycoplasmas and especially M. fermentans in CFS patients.

A polymerase chain reaction (PCR)-based assay was used to detect Mycoplasma genus and M. fermentans genomes in peripheral blood mononuclear cells (PBMC) of CFS patients. Blood was collected from 100 patients with CFS and 50 control subjects. The amplified products of 717 bp of Mycoplasma genus, and 206 bp of M. fermentans were detected in DNA purified from blood samples in 52% and 34% of CFS samples, respectively. In contrast, these genomes were found in only 14% and 8% of healthy control subjects respectively (P < 0.0001).

All samples were confirmed by Southern blot with a specific probe based on internal sequences of the expected amplification product. Several samples, which were positive for Mycoplasma genus, were negative for M. fermentans indicating that other Mycoplasma species are involved. A quantitative PCR was developed to determine the number of M. fermentans genome copies present in 1 microg of DNA for controls and CFS patients.

Mycoplasma copy numbers ranging from 130 to 880 and from 264 to 2400 were detected in controls and CFS positive subjects, respectively. An enzyme immunoassay was applied for the detection of antibodies against p29 surface lipoprotein of M. fermentans to determine the relationship between M. fermentans genome copy numbers and antibody levels. Individuals with high genome copy numbers exhibited higher IgG and IgM antibodies against M. fermentans specific peptides. Isolation of this organism by culture from clinical specimens is needed in order to demonstrate specificity of signal detected by PCR in this study.

 

Source: Vojdani A, Choppa PC, Tagle C, Andrin R, Samimi B, Lapp CW. Detection of Mycoplasma genus and Mycoplasma fermentans by PCR in patients with Chronic Fatigue Syndrome. FEMS Immunol Med Microbiol. 1998 Dec;22(4):355-65. http://femsim.oxfordjournals.org/content/22/4/355.long (Full article)

 

Elevated apoptotic cell population in patients with chronic fatigue syndrome: the pivotal role of protein kinase RNA

Abstract:

OBJECTIVES: A prominent feature of chronic fatigue syndrome (CFS) is a disordered immune system. Recent evidence indicates that induction of apoptosis might be mediated in a dysregulated immune system by the upregulation of growth inhibitory cytokines. Therefore, the purpose of this study was to evaluate the apoptotic cell population, interferon-alpha (IFN-alpha) and the IFN-induced protein kinase RNA (PKR) gene transcripts in peripheral blood lymphocytes (PBL) of CFS individuals, as compared to healthy controls.

SUBJECTS AND METHODS: PBL were isolated from CFS (n = 29) and healthy control individuals (n = 15) and subjected to quantitative analysis of apoptotic cell population and cell cycle progression by flow cytometry. Quantitative competitive polymerase chain reaction (Q/C PCR) and Western blot analysis were used to assess the levels of PKR mRNA and protein in control and CFS individuals. In addition, circulating IFN-alpha was measured by ELISA assay.

RESULTS: Increased apoptotic cell population was observed in CFS individuals, as compared to healthy controls (26.6 +/- 12.9% and 9.9 +/- 4.2%, respectively). The increased apoptotic subpopulation in CFS individuals was accompanied by an abnormal cell arrest in the S phase and the G2/M boundary of the cell cycle as compared to the control group (8.6 +/- 1.2 to 22.8 +/- 2.4 and 3.6 +/- 0.82 to 24.3 +/- 3.4, respectively). In addition, CFS individuals exhibited enhanced PKR mRNA and protein levels (mean basal level 3538 +/- 1050 and 2.7 +/- 0.26, respectively) as compared to healthy controls (mean basal level 562 +/- 162 and 0.89 +/- 0.18, respectively). In 50% of the CFS samples (n = 29) treated with 2-aminopurine (2-AP) (a potent inhibitor of PKR) the apoptotic population was reduced by more then 50%.

CONCLUSIONS: PKR-mediated apoptosis in CFS individuals may contribute to the pathogenesis and the fatigue symptomatology associated with CFS.

Comment in: Cortisol deficiency may account for elevated apoptotic cell population in patients with chronic fatigue syndrome. [J Intern Med. 1999]

 

Source: Vojdani A, Ghoneum M, Choppa PC, Magtoto L, Lapp CW. Elevated apoptotic cell population in patients with chronic fatigue syndrome: the pivotal role of protein kinase RNA. J Intern Med. 1997 Dec;242(6):465-78. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2796.1997.tb00019.x/epdf (Full article)