Deficiency in the Expression of STAT1 Protein in a Subpopulation of Patients with Chronic Fatigue Syndrome (CFS)
Status:
Archived
Authors:
Alan Cocchetto
Date Published:
2004
Dr. Konstance Knox Presents STAT1 Data to
Researchers and Clinicians at AACFS Conference
by Alan Cocchetto, NCF Medical Director (Copyright 2004)
The American Association for Chronic Fatigue Syndrome (AACFS) held its latest conference
on October 8-10, 2004 in Madison, Wisconsin. The three-day conference was co-sponsored
by the Centers for Disease Control (CDC), the National Center for Infectious Diseases and
hosted locally by the Wisconsin CFS Association.
on October 8-10, 2004 in Madison, Wisconsin. The three-day conference was co-sponsored
by the Centers for Disease Control (CDC), the National Center for Infectious Diseases and
hosted locally by the Wisconsin CFS Association.
The National CFIDS Foundation (NCF) sponsored this STAT1 study through its Research Grant Program with sole funding provided by patient donations. STAT1 is known to be a critically important protein necessary for mounting an appropriate immune response to bacterial and viral pathogens. Details of its importance in host immunity has been previously reported upon in an investigative report in the Forum. Click here to read our articles about STAT1.
This research project was completed by Dr. Konstance Knox and Dr. Donald Carrigan, scientists from the Institute for Viral Pathogenesis (IVP). At the AACFS Conference, Dr. Konstance Knox presented the following research data in the microbiology/immunology portion of the conference:
"Deficiency in the Expression of STAT1 Protein in a Subpopulation
of Patients with Chronic Fatigue Syndrome (CFS)"
by KK Knox, A Cocchetto, E Jordan, D Leech, DR Carrigan
Background:
CFS is a debilitating illness associated with persistent severe fatigue, a variety of physical and
neuropsychological signs and symptoms, and frequently, an unusual susceptibility to a variety
of infections. The study of intracellular signal transduction pathways may have provided a key
insight into the immunological defect operative in CFS patients. Signal transducers and activators of transcription (STAT) are a family of proteins that play central roles in the responses of cells to cytokines. Specifically, the protein STAT1 is intimately involved in the response of cells to Type I (alpha and beta) and Type II (gamma) interferons.
CFS is a debilitating illness associated with persistent severe fatigue, a variety of physical and
neuropsychological signs and symptoms, and frequently, an unusual susceptibility to a variety
of infections. The study of intracellular signal transduction pathways may have provided a key
insight into the immunological defect operative in CFS patients. Signal transducers and activators of transcription (STAT) are a family of proteins that play central roles in the responses of cells to cytokines. Specifically, the protein STAT1 is intimately involved in the response of cells to Type I (alpha and beta) and Type II (gamma) interferons.
Objectives:
The aim of these studies was to evaluate samples of peripheral blood mononuclear cells (PBMC) from CFS patients and healthy control subjects for the expression of STAT1.
The aim of these studies was to evaluate samples of peripheral blood mononuclear cells (PBMC) from CFS patients and healthy control subjects for the expression of STAT1.
Methods:
PBMC were purified and lysed in a buffer containing non-ionic detergent, a protease inhibitor cocktail and a combination of two proteasomal protease inhibitors (MG-132 and lactacystin). Samples corresponding to known numbers of PBMC were then subjected to an immunoblotting procedure using a STAT1 specific antiserum (sc-592; Santa Cruz Biotechnology). A quantitation method was developed using densitometric scanning of the protein bands detected by the antiserum. Titration experiments with PBMC from control subjects demonstrated that STAT1 protein detection was quantitatively linear for samples of between 20000 and 140000 cells per well in PAGE. Therefore, PBMC samples were screened for STAT1 protein using 100000 cells per well. Adequacy of protein in all samples was monitored by staining the immunoblots for actin.
PBMC were purified and lysed in a buffer containing non-ionic detergent, a protease inhibitor cocktail and a combination of two proteasomal protease inhibitors (MG-132 and lactacystin). Samples corresponding to known numbers of PBMC were then subjected to an immunoblotting procedure using a STAT1 specific antiserum (sc-592; Santa Cruz Biotechnology). A quantitation method was developed using densitometric scanning of the protein bands detected by the antiserum. Titration experiments with PBMC from control subjects demonstrated that STAT1 protein detection was quantitatively linear for samples of between 20000 and 140000 cells per well in PAGE. Therefore, PBMC samples were screened for STAT1 protein using 100000 cells per well. Adequacy of protein in all samples was monitored by staining the immunoblots for actin.
Results:
In both CFS patients and controls, five proteins reacting with the sc592 antiserum were identified. Specificity of the antiserum staining was demonstrated by complete blocking of the detection of all five proteins by preincubation of the antiserum with the peptide used for production of the antiserum. STAT1 proteins detected were (1) a minor, inconsistent band of approximately 150 kiloDaltons (kD) (function unknown), (2) a strong band at 91 kD (STAT1-alpha splice variant; STAT1-91), (3) a strong band at 84 kD (STAT1-beta splice variant; STAT1-84), (4) a strong band at 56 kD (function unknown; STAT1-56) and (5) a strong band at 51 kD (function unknown; STAT1-51). STAT1-91 and STAT1-84 were not consistently resolved from one another and were analysed together (STAT1-91/84). Results based on 27 controls and 25 CFS patient samples were presented at the conference on several overheads as follows:
In both CFS patients and controls, five proteins reacting with the sc592 antiserum were identified. Specificity of the antiserum staining was demonstrated by complete blocking of the detection of all five proteins by preincubation of the antiserum with the peptide used for production of the antiserum. STAT1 proteins detected were (1) a minor, inconsistent band of approximately 150 kiloDaltons (kD) (function unknown), (2) a strong band at 91 kD (STAT1-alpha splice variant; STAT1-91), (3) a strong band at 84 kD (STAT1-beta splice variant; STAT1-84), (4) a strong band at 56 kD (function unknown; STAT1-56) and (5) a strong band at 51 kD (function unknown; STAT1-51). STAT1-91 and STAT1-84 were not consistently resolved from one another and were analysed together (STAT1-91/84). Results based on 27 controls and 25 CFS patient samples were presented at the conference on several overheads as follows:
Two-Sided Fisher's Exact Test Analysis of STAT1-91/84 Expression
in Healthy Control Subjects and CFS Patients:
| Healthy Controls | CFS Patients | Total | |
| STAT1-91/84 Negative |
1 (4%) |
8 (32%) |
9 |
| STAT1-91/84 Positive |
26 (96%) |
17 (68%) |
43 |
| Total | 27 (100%) |
25 (100%) |
52 |
p = 0.0098; Very Significant
Comparison of Positivity for STAT-91/84, STAT1-56 and STAT1-51 for
Healthy Control Subjects and CFS Patients:
| Subjects | Percent STAT1-91/84 Positive |
Percent STAT1-56 Positive |
Percent STAT1-51 Positive |
| CFS Patients | 68% (17/25) |
84% (21/25) |
100% (25/25) |
| Healthy Controls | 96% (26/27) |
93% (25/27) |
96% (26/27) |
Discussion:
Dr. Knox's overheads included the following discussion highlights that were presented to the
audience at her presentation:
Dr. Knox's overheads included the following discussion highlights that were presented to the
audience at her presentation:
* STAT is known as Signal Transducers and Activators of Transcription
- Family of transcription factors
- Activated by a variety of cytokines including:
Interferons: alpha-interferon, beta-interferon and gamma-interferon
Interleukins: IL-2,3,4,5,6,7,9,10,12,13,15
Growth hormones: erythropoietin/EPO, growth hormone/GH, prolactinPRL
Growth factors: EDF, PDGF, CSF-1, G-CSF
- Family of transcription factors
- Activated by a variety of cytokines including:
Interferons: alpha-interferon, beta-interferon and gamma-interferon
Interleukins: IL-2,3,4,5,6,7,9,10,12,13,15
Growth hormones: erythropoietin/EPO, growth hormone/GH, prolactinPRL
Growth factors: EDF, PDGF, CSF-1, G-CSF
* STAT: Involved in a wide spectrum of physiological responses
- Cell proliferation
- Cell differentiation
- Apoptosis
- Immune responses
- Development
- Hematopoiesis
- Cell proliferation
- Cell differentiation
- Apoptosis
- Immune responses
- Development
- Hematopoiesis
* STAT1:
- Centrally involved in the response of cells to:
Type 1 interferons (alpha and beta)
Type 2 interferons (gamma)
- In both animals and humans, defects in STAT1 are associated with fatal infections
by both viruses and bacteria
- Centrally involved in the response of cells to:
Type 1 interferons (alpha and beta)
Type 2 interferons (gamma)
- In both animals and humans, defects in STAT1 are associated with fatal infections
by both viruses and bacteria
* Importance of STAT1 in CFS stems from recent observations suggesting that Type 1
interferon responses are abnormal in many patients with CFS
* An interesting observation in this area of research is that of Englebienne and colleagues:
- As the RNase L ratio (37 kD/80 kD) increases, you see a decrease in STAT1 protein
- Hypothesis: STAT1 is cleaved/degraded by the same enzyme that is cleaving RNase L
- As the RNase L ratio (37 kD/80 kD) increases, you see a decrease in STAT1 protein
- Hypothesis: STAT1 is cleaved/degraded by the same enzyme that is cleaving RNase L
* IVP study of STAT1 protein expression in CFS patients:
- Blood samples from 25 CFS patients were studied from two physician based practices
in Rochester, NY and Albuquerque, NM
- Blood samples from 27 healthy control subjects were obtained from laboratory
personnel or purchased from Analytical Biological Sciences Inc, Wilmington DE
- Blood samples from 25 CFS patients were studied from two physician based practices
in Rochester, NY and Albuquerque, NM
- Blood samples from 27 healthy control subjects were obtained from laboratory
personnel or purchased from Analytical Biological Sciences Inc, Wilmington DE
* IVP study of STAT1 protein expression in CFS patients:
- Upon receipt in the laboratory by overnight courier, PBMC were purified by
centrifugation and known numbers of cells were frozen as cell pellets at -70 degrees C.
- Immediately before use, several protease inhibitors were added to the lysis buffer
- Two proteasomal protease inhibitors were also added immediately before use of the
lysis buffer
- STAT1 proteins were detected on the membranes by means of an antiserum specific for
an epitope in the carboxy terminus of STAT1 (sc-592)
- Upon receipt in the laboratory by overnight courier, PBMC were purified by
centrifugation and known numbers of cells were frozen as cell pellets at -70 degrees C.
- Immediately before use, several protease inhibitors were added to the lysis buffer
- Two proteasomal protease inhibitors were also added immediately before use of the
lysis buffer
- STAT1 proteins were detected on the membranes by means of an antiserum specific for
an epitope in the carboxy terminus of STAT1 (sc-592)
* IVP study conclusions:
- STAT1 is comprised of at least five antigenically related proteins with the two most
fully studied being the STAT1-91 (alpha splicing variant) and STAT1-84 (beta splicing
variant).
- A subset (approximately 30%) of patients with CFS have a quantitative deficiency in
the expression of STAT1-91/84 proteins.
- Another subset of patients with CFS were found to have abnormally high levels of
STAT1-91/84 proteins.
- The STAT1 deficiency may predispose the patients to develop a variety of infections
due to dysfunction of the Type 1 and Type 2 interferon systems.
- STAT1 is comprised of at least five antigenically related proteins with the two most
fully studied being the STAT1-91 (alpha splicing variant) and STAT1-84 (beta splicing
variant).
- A subset (approximately 30%) of patients with CFS have a quantitative deficiency in
the expression of STAT1-91/84 proteins.
- Another subset of patients with CFS were found to have abnormally high levels of
STAT1-91/84 proteins.
- The STAT1 deficiency may predispose the patients to develop a variety of infections
due to dysfunction of the Type 1 and Type 2 interferon systems.
The NCF comments on the study:
Gail Kansky, President of the NCF, stated "This research study formally establishes the extent of the immune dysfunction, through our quantification of the STAT1 protein, in Chronic Fatigue and Immune Dysfunction Syndrome - CFIDS. To find that approximately 30% of our patients tested in this study had little or no STAT1 protein is highly significant. We are most appreciative to Dr. Konstance Knox and Dr. Donald Carrigan for their scientific efforts in this study; to Dr. Edward Jordan and Dr. David Leech who participated in this study; and to the patients who were personally involved with the study themselves. Furthermore, without the financial support from our patient community that strongly believed in these research efforts, this study never would have gotten off the ground" said Kansky. "Let's face it, STAT1 deficiency isn't malingering nor is it psychological! This immune problem is very real and it is certainly very serious" stated Kansky.
Gail Kansky, President of the NCF, stated "This research study formally establishes the extent of the immune dysfunction, through our quantification of the STAT1 protein, in Chronic Fatigue and Immune Dysfunction Syndrome - CFIDS. To find that approximately 30% of our patients tested in this study had little or no STAT1 protein is highly significant. We are most appreciative to Dr. Konstance Knox and Dr. Donald Carrigan for their scientific efforts in this study; to Dr. Edward Jordan and Dr. David Leech who participated in this study; and to the patients who were personally involved with the study themselves. Furthermore, without the financial support from our patient community that strongly believed in these research efforts, this study never would have gotten off the ground" said Kansky. "Let's face it, STAT1 deficiency isn't malingering nor is it psychological! This immune problem is very real and it is certainly very serious" stated Kansky.
During this interview, Kansky went on to say that "The National CFIDS Foundation is most confident in our research direction! We have several studies that are currently underway and these reflect the NCF's absolute conviction to conquer this disease. This additional research work should bring important new knowledge to our understanding of this devastating disease while offering true hope to those who suffer from it. In addition, we are anticipating the publication of these research studies in peer-reviewed medical journals. This is a vital step
in acknowledging the severity of this disease in the medical community."
in acknowledging the severity of this disease in the medical community."
Additional importance of STAT1 cannot be understated:
The latest research helps us to understand the underlying importance and seriousness of STAT1 with regards to the immune system response to infections. Researchers in the U.S. have now shown that animals that lack the STAT1 protein lack the ability to restrain or restrict infections in cells and tissues. Stated another way, ordinary infections, such as influenza, that normally only affect lung tissue cannot restrict the infection to only lung tissue in STAT1 deficient hosts - other cells and tissues subsequently become permissive to infection so that an influenza or other common infections could now infiltrate other organs systemically. As you can now understand and appreciate, the lack of STAT1 is a severe host defect. This inherent lack of restriction implies that secondary or tertiary infections could potentially overtake the body's own defense system due to the lack of STAT1 protein. Thus, these other infections would act to throw scientific researchers "off the track" of the true culprit/agent primarily responsible for damaging the immune system in such an elusive and covert manner! Therefore, the importance of this mechanism, that is operative in this disease, cannot be understated nor underestimated and must be one that is carefully studied scientifically. This is why the NCF is fully committed to this research effort. We believe that true hope lies within the medical truths of this disease and that our blood is leading us to the truth....now, we just have to listen!
The latest research helps us to understand the underlying importance and seriousness of STAT1 with regards to the immune system response to infections. Researchers in the U.S. have now shown that animals that lack the STAT1 protein lack the ability to restrain or restrict infections in cells and tissues. Stated another way, ordinary infections, such as influenza, that normally only affect lung tissue cannot restrict the infection to only lung tissue in STAT1 deficient hosts - other cells and tissues subsequently become permissive to infection so that an influenza or other common infections could now infiltrate other organs systemically. As you can now understand and appreciate, the lack of STAT1 is a severe host defect. This inherent lack of restriction implies that secondary or tertiary infections could potentially overtake the body's own defense system due to the lack of STAT1 protein. Thus, these other infections would act to throw scientific researchers "off the track" of the true culprit/agent primarily responsible for damaging the immune system in such an elusive and covert manner! Therefore, the importance of this mechanism, that is operative in this disease, cannot be understated nor underestimated and must be one that is carefully studied scientifically. This is why the NCF is fully committed to this research effort. We believe that true hope lies within the medical truths of this disease and that our blood is leading us to the truth....now, we just have to listen!
