Part One - Unmasked Research: STAT1-alpha and p53 Deficiencies are Found in Patients with Chronic Fatigue Syndrome; The National CFIDS Foundation Responds by Funding Several New Research Initiatives

Investigative Report by Alan Cocchetto, Medical Director, NCF, Inc.   
© 2003 Written Permission Required for Reprinting or Distributing

Through its own extensive six month medical investigation, the National CFIDS Foundation (NCF) uncovered research that will have widespread ramifications among the CFS patient community, their physicians, as well as CFS researchers worldwide establishing CFS as a serious and perhaps fatal illness. Because of these implications, the NCF has responded to this major discovery by dynamically funding new medical research by several noted scientists.
 
Briefly stated, the NCF has uncovered a key scientific discovery, previously made by prominent CFS researchers, that has yet to see the 'light of day.' The critical component: STAT1. The STAT1 protein is crucial for proper immune function and regulation. Without it, cells are unresponsive to interferons leaving the body defenseless against viral and bacterial infections. In fact, human STAT1 deficiency is lethal. In this report, the NCF identified CFS researchers that previously found STAT1 to be absent in CFS patient blood.

"The NCF began its investigation by thoroughly reviewing work that had been previously published by Kenny DeMeirleir, MD, PhD and his colleague's group in Belgium" stated Gail Kansky, President of the NCF. "Then one discovery lead to another and our own investigation just skyrocketed" said Kansky. "As the CFS patient community is aware, Dr. DeMeirleir is a highly visible CFS researcher, both in the U.S. and abroad. He is a medical editor for the Journal of Chronic Fatigue Syndrome, is on the board of the American Association for Chronic Fatigue Syndrome (AACFS), and is actively involved with R.E.D. Laboratories which has worked to further the studies of Dr. Robert Suhadolnik, from Temple University, whose research group made the initial RNase L discoveries in CFS patients" stated Kansky. "I strongly feel that the NCF's investigational and medical research efforts will greatly add to those efforts that we have previously undertaken to unravel the mysteries of CFS. This should lead to much greater understanding of the disease process" said Kansky. Furthermore, she stated that "Our funded medical research is aimed to solidify and scientifically expand this important work so that desperately needed and appropriate patient treatments will follow. The NCF's efforts will continue to illuminate the darkness that has overshadowed this very serious illness."

The NCF first began its investigation with a published book titled Chronic Fatigue Syndrome: A Biological Approach edited by Dr. Patrick Englebienne and Dr. Kenny DeMeirleir [1]. Published in 2002, examination of the chapters, by NCF staffers, revealed that Dr. DeMeirleir and his colleagues carefully and methodically probed various signal transduction pathways in patients with CFS via examination of their peripheral blood mononuclear cells (PBMC's or monocytes). These authors stated that "the importance of STAT1 in mediating the action of both type I and II IFNs (interferons) is no longer questioned, as a lack of its expression is consistently associated with IFN resistance. The apparent dysregulation in types I and II IFN pathways in chronic fatigue syndrome led us to investigate the expression of STAT1 in PBMCs. We classified the samples according to the ratio of 37- over 80- kDa RNase L which is representative of the proteolytic activity of the PBMC samples. As shown in Figure 5.9, STAT1 is fully degraded in positive samples, suggesting that it may also be a substrate of the proteases responsible for RNase L cleavage. A degradation of STAT1 in those cells might well constitute the missing link explaining unresponsiveness to IFNs" (interferons) [2].

In this figure from the book, with RNase L ratios (37 kDa/80 kDa) greater than 0.25, native STAT1-alpha is lost but some cleavage (breakdown) products are shown until the RNase L ratio hits approximately 2.0. After that, there is the complete absence of both STAT1-alpha and the cleavage product! Explaining further, STAT is notation for Signal Transducers and Activators of Transcription. STAT's are a family of transcription factors that play central roles in the responses of cells to cytokines, molecules that control every aspect of the immune system [3]. STAT1 has two forms, alpha and beta. Dr. DeMeirleir's group tested STAT1-alpha, a native 91 kDa protein, in patients with CFS and correlated it with the RNase L ratios. STAT1-alpha is intimately involved in the response of cells to type I (alpha and beta) and type II (gamma) interferons. Most important is the fact that a STAT1-alpha deficiency is associated with fatal infections by both viruses and bacteria [3,4,5,6,7,8]. Furthermore, in animals, STAT1-alpha deficiency is associated with impaired responses to interferons, increased susceptibility to tumors, as well as impaired growth control. STAT1-alpha also plays a critically important role in antigen presentation.

The NCF staff then wondered if Dr. DeMeirleir and his colleagues realized the importance, as well as the significance, of their discovery and when might they have first identified it? To answer this question, we uncovered a patent filed in April 1999 titled "Methods and compositions for use in characterizing multiple sclerosis disease activity in a subject." [9] In this patent, Dr. DeMeirleir and his colleagues at R.E.D. Laboratories determined the amount of 37 kDa and 80 kDa RNase L proteins and utilized RNase L ratios to characterize multiple sclerosis disease activity. This patent was filed after one filed by inventor Dr. Robert Suhadolnik, in April 1999, titled "Chronic fatigue syndrome diagnosis" in which the diagnosis of CFS was made through the detection and determination of both the 37 kDa and 80 kDa RNase L proteins [10]. Examination of both these patents indicated that, in both CFS and MS, these same RNase L proteins can be used to assist in diagnosis as well as for determining disease activity. Multiple sclerosis patients have previously been found to have alterations in STAT1-alpha [11]. Furthermore, in CFS, this is vitally important due to the correlations that were found between the RNase L ratios and STAT1-alpha.

This is further stated in two additional patent applications by Dr. DeMeirleir's colleagues. One of these applications directly discusses the specific role of STAT1-alpha in CFS patients [12,13]. Quoting these patent applications directly: "STAT1 plays an important role in growth arrest, in promoting apoptosis and is implicated as a tumor suppressor. STAT1 null cells are resistant to apoptotic induction by TNF-alpha....STAT1 deficient mice exhibit a severe defect in IFN-dependent immune responses against viruses and microbial pathogens....If STAT1 is disabled or otherwise inactive in the cells of the immune system, treatment with interferon or interferon inducer will not be effective in promoting and establishing the interferon-inducible antiviral and antiproliferative pathways." Furthermore these applications state "the results demonstrate that the presence and amount of STAT1 protein fragmentation directly correlates with the presence and amount of low molecular weight RNase L fragments in PBMC samples. These data indicate that native STAT1 protein is fragmented at an earlier point in the disease cycle than RNase L, and that by the time native RNase L is demonstrably attacked by proteases (ratio > 2.0), that native STAT1 protein has entirely disappeared due to proteolysis, leaving the cells unable to respond to interferons and/or interferon inducers."

It became obvious to NCF staffers that Dr. DeMeirleir and his colleagues had made a vitally important discovery but had only published part of the information in the book and the remainder in patents or patent applications that we were aware of. Was there other information to be found?

A quick check of the R.E.D. Laboratories website [14] indicated that "The company's scientists have discovered a number of other cellular proteins that play important roles in the initiation, progression, and pathogenesis of immune dysfunction. The most important revelation to date has been the discovery of one protein, which is used as a serum-based assay for the detection of chronic immune disease. Such a serum-based marker has been developed as a screening test for the blood supply to reduce the number of transfusion-related infections with persistent (and covert) viral infections."

It became obvious to the NCF staffers, that both the book and the patent information told a significant story regarding the critical importance of STAT1-alpha in CFS patient blood. One thing is certain, blood doesn't lie! Likewise, the true implications, pathological and perhaps life threatening for patients as well as financial potentially for R.E.D. Laboratories, regarding transfusion-related infections in the blood supply by "covert infections", were enormous and certainly worthy of worldwide attention!

Probing further, the NCF discovered vitally important testimony given directly by Dr. Kenny DeMeirleir before the Flemish Parliament of Belgium in a hearing on March 5, 2001 [15]. In his testimony given before Flemish Parliament, Dr. DeMeirleir stated "Caspases and calpain are induced by cellular stress, which leads to apoptosis. In an intracellular disorder, calcium influx is increased. Calcium will further activate calpain, so that some caspases are inhibited and therefore block apoptosis. One of the cellular proteins that are split by these enzymes is STAT1, the carrier of the interferon-gamma signal in the immune cells, which leads to the Th1-to-Th2 shift. Unfortunately, the Chronic Fatigue Syndrome is often called psychosomatic. This is, however, more indicative of medicine's inability to deal with it. We now understand the nature of the disorder. In its early phase, apoptosis increases. In a subsequent evolution, apoptosis is blocked and the interferon signal disappears due to the destruction of the protein which transports the intracellular interferon signal to the nucleus (where apoptosis is initiated). This leads to more and more infections. This process goes on at all levels (in the central nervous system, the muscle cells, the white blood cells, and so on). Some patients suffering from the Chronic Fatigue Syndrome develop epilepsy. We find that most patients have a light form of epilepsy. This leads to sleep disturbances and a situation where the fatigue increases since one does not recover anymore."
"Given the true significance of this work and its implication in a functionally oppressive disease that desperately struggles for proper recognition, validation, and affirmation at all levels of medical science, the NCF's Board of Directors unanimously voted to immediately fund several important research grant projects to be carried out by noted researchers" said Kansky.

The NCF first contacted a well-known research group, who has asked to remain anonymous at this time, and arranged to send grant funding to them via our NCF Research Grant Program. "We are very excited by this since this group will be the first to begin studying "STAT1-alpha in Chronic Fatigue Syndrome Patients" stated Kansky. Lead scientists for this research group commented that this was an enormous step forward towards understanding the disease process in these patients.

Next, the NCF arranged another research project, also funded by our Research Grant Program. The NCF sent grant money to Dr. Konstance Knox and Dr. Donald Carrigan, both from the Institute for Viral Pathogenesis, for "A Study for the Potential Role of STAT1 in the Pathogenesis of Chronic Fatigue Syndrome." "This grant is aimed at significantly expanding our knowledge about the specifics of STAT1 and its role in CFS from a pathological standpoint" stated Kansky. "Futhermore, since we are expecting both teams (first group and Knox/Carrigan) to complete their work with research physicians in the months ahead, we are anticipating significant medical breakthroughs as the direct result of these grants. The NCF has expedited this work because our patients are seriously ill and we anticipate that these efforts will certainly confirm this" said Kansky.
The NCF staff also contacted two world renown experts on the STAT1 protein: Dr. Joan Durbin from Columbus Children's Research Institute and Dr. David Levy from New York University School of Medicine. Both Dr. Durbin and Dr. Levy have published extensively on STAT1 and are responsible for the development of the animal model for STAT1-alpha deficiencies. In NCF interviews with both of them and from their numerous published medical journal articles, it is clear that the complete loss of the native STAT1 protein constitutes a serious illness that may ultimately be fatal unless this protein defect can be corrected.
Ironically, this is where the NCF is truly hopeful. In one of the patent applications [13], Dr. DeMeirleir's colleagues determined that the "STAT1 protein is degraded when a cell extract from a healthy control (i.e., 'negative extract'; RNase L ratio < 0.2, STAT1 protein containing) is incubated with a cell extract from a patient (RNase L ratio = 3.0; STAT1 protein negative). This degradation is inhibited in the presence of proteasomal inhibitor (MG132) but not in the presence of the other protease inhibitors tested. Thus the degradation of STAT1 protein is a specific cellular process that involves proteasome and does not involve the apoptotic enzymes caspase-3 or calpain."

What this implies is that Dr. DeMeirleir's colleagues had already found a very specific inhibitor, MG132, for STAT1-alpha degradation in CFS patient blood in-vitro! In our own research, the NCF found that MG132 functions as both a specific inhibitor of the ubiquitin-proteasome pathway [16] as well as a specific beta-secretase inhibitor [17]. Both of these pathways are intimately involved with amyloid formation. Amyloidogenesis is a general phenomenon of protein misfolding. Protein misfolding and the formation of abnormal protein fibrils appear to play a central role in a variety of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. This is why the expression of the Huntington's protein in CFS patients, by the CDC using gene expression analysis, is intriguing [18]. These are all scientific clues to a bigger picture for this disease.

Because of our previous efforts with the ciguatera epitope and its effects on neuroblastoma cells as well as other specific unannounced scientific findings related to this epitope, the NCF has also provided new research grant funding to Dr. Yoshitsugi Hokama, from the University of Hawaii, for the "Development of an Immunological Assay for Assessment of Amyloids in Chronic Fatigue Syndrome." "Dr. Hokama's previous research dovetailed nicely into the scientific work we are describing here" said Kansky. "In fact, the NCF believes there is a relationship between the ciguatera epitope, protein misfolding, and amyloid formation.

Amyloids directly alter sodium channels and that is part of the significance of the ciguatera epitope finding as it relates to our previous CFS patient studies. This is something that we had known prior to the initial funding of Dr. Hokama's work but this previous work had to be scientifically verified and expanded upon first before we pressed on further" stated Kansky. The NCF is also aware that a relationship already exists between amyloid formation and STAT1-alpha. Amyloids can potentially bind the STAT1-alpha protein and take it out of commission. This mechanism is very important. Dr. DeMeirleir and his colleague's discovery on STAT1-alpha and RNase L cleavage is central to our working hypothesis but the NCF believes that we have found other missing pieces, regarding the infectious agent as well as other disease mechanisms, that are absent from their work. What is exciting is that our NCF staff has already identified very specific PCR binding sequences for STAT1-alpha in the infectious agent that we believe is directly responsible for this disease. This implies that once infection begins, STAT1-alpha becomes one of the first critical immune system proteins to get "chewed up" as a result of being a direct target of the infectious agent thereby greatly damaging the immune system! This would be in agreement with Dr. De Meirleir's findings where STAT1-alpha binding occurs before the degradation of the native RNase L protein.

"Furthermore, in full agreement with Dr. Elaine DeFreitas' previous work, the NCF believes this infectious agent is of non-human origin" stated Kansky. "We are meeting the dragon head-on and we hope that our funded research will help crack the code, once and for all, for this disease. The NCF continues to break important new ground in the understanding of the disease mechanisms while going scientifically where no other CFS organization has ever been before! This appears to be our forte and it reflects our true passion and full committment to the CFS patient community. As a result, we are much further ahead in the discovery process than most patients and researchers realize" said Kansky.

In addition, Dr. DeMeirleir's colleagues also made another significant discovery critical to the health and welfare of patients with CFS [19,20]. Excerpts from the book and the patent application state "Fig. 1 represents a densitometric scan of a Western blot detecting p53 protein and p53 protein fragments from PBMC extracts from CFS patients....The above results demonstrate that the presence and amount of p53 protein fragmentation directly correlates with the presence and amount of low molecular weight RNase L fragments in PBMC samples. These data indicate that native p53 protein is fragmented at a later point in the disease cycle than RNase L protein. The loss of functional p53 protein in PBMCs render these cells unable to respond to normal growth inhibitory stimuli and provide the means whereby unregulated cell growth occurs, ultimately giving rise to hematopoietic tumors."

Furthermore, their explanation in this application states: "Another important protein that regulates the induction of apoptosis is p53. The p53 protein is normally activated in response to genetic damage within the cell and its activation is accompanied by self-stabilization, allowing it to accumulate to high levels and cause cell cycle arrest and induce apoptosis. In addition, the p53 protein has a critical role in protecting the cell from malignant development; mutations in the p53 gene (and protein) are the most frequently detected genetic event in cancer.
Mutations in p53 may occur at the genetic level (i.e. DNA sequence alterations that change the amino acid structure of the protein), or its function may be altered by alterations in the numerous proteins with which p53 interacts. p53 may also be altered by the action of certain proteases, resulting in cleavage, preventing the formation of active tetramers of the protein. If p53 is cleaved and/or otherwise disabled in the cells of the immune system, these cells would be blocked from entering the apoptotic pathway if infected with a virus or other microorganism. In addition, persistent inactivation of the p53 protein may lead to increased incidence of cancer (Levine et. al., J. Chronic Fatigue Syndrome 7: 29-38, 2000). Activation of the 2-5A synthetase / RNase L antiviral pathway has been demonstrated to induce apoptosis, while induction of the same pathway in cells expressing mutant forms of p53 was demonstrated to suppress the apoptotic pathway. The inactivation of p53, RNase L, and other proteins within the cells of the immune system most certainly leads to a dysfunctional immune system, unable to respond to challenge by microorganisms and/or the presence of pre-malignant cells.

Indeed the immune system itself may be in a pre-malignant state."
In a review of the literature on STAT1-alpha and p53, we found an article [21] on STAT1 deficient mice that states "when the STAT1 deficiency was placed on a p53 null background there were increased rates of tumor formation and an increase in the non-lymphoid tumor types." This helps to explain the importance of these two proteins that are essential to appropriate immune responses since STAT1 and interferon are central to antitumor effects [22]. In fact, this study suggests that NK (Natural Killer) cells from STAT1 deficient mice may have a reduced capacity to eliminate tumor cells in-vivo.

Part one article review at a glance:

Dr. DeMeirleir and colleagues scientific finding:
STAT1-alpha deficiency in CFS patients
p53 deficiency in CFS patients

Scientific interpretation:
Loss of STAT1-alpha constitutes a serious illness that may ultimately be fatal because the cells are unresponsive to interferon leaving them unable to adequately defend against infections.

Loss of p53 constitutes a pre-malignant state because surveillance against DNA mutations, protein alterations, and unregulated cell growth/division are left unguarded.

Loss of these proteins assists in immune deficiency and dysregulation.
 
[Ed. Note: The references for this article have been combined with the article, "Total Exposure."]