Abstract
Presented at the Seventh International AACFS
Conference on Chronic Fatigue Syndrome, Fibromyalgia and other
Related Illnesses. Madison,
Wisconsin; October, 2004
DEFICIENCY IN THE EXPRESSION OF STAT1 PROTEIN IN A SUBPOPULATION
OF PATIENTS WITH CHRONIC FATIGUE SYNDROME (CFS).
KK Knox1,
A Cocchetto2,
E Jordan3,
D Leech4
and DR Carrigan1.
1
Institute for Viral Pathogenesis; Milwaukee, WI; 2National
CFIDS Foundation; Needham, MA; 3
Olean, NY, 4
Albuquerque; NM.
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.
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.
Methods.
PBMC were purified and lysed in a buffer containing non-ionic
detergent, a protease inhibitor cocktail and a combination of two
proteosomal 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 2 X 104
and 1.4 X 105
cells per well in PAGE. Therefore,
PBMC samples were screened for STAT1 protein using 105
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 91kD (STAT1 alpha splice variant; STAT1-91), (3) a strong
band at 84kD (STAT1 beta splice variant; STAT1-84), (4) a strong
band at 56kD (function unknown; STAT1-56) and (5) a strong band at
51kD (function unknown; STAT1-51).
STAT1-91 and STAT1-84 were not consistently resolved from one
another and were analysed together (STAT1-91/84).
Results are summarized below:
|
Subject
Group
|
STAT1-91/84
+ STAT1-56
+
STAT1-51+
|
STAT1-91/84
-
STAT1-56
+
STAT1-51
+
|
STAT1-91/84
-
STAT1-56
-
STAT1-51
+
|
STAT1-91/84
+
STAT1-56
-
STAT1-51
-
|
|
Controls
|
92%
(25/27)
|
16%
(4/25)
|
16%
(4/25)
|
0%
(0/25)
|
|
CFS
Patients
|
68%
(17/25)
|
0%
(0/27)
|
4%
(1/27)
|
4%
(1/25)
|
Conclusions.
STAT-1-91/84 represents the form of STAT1 that is involved in
the intracellular signaling of both type I and type II interferons. The higher negativity for STAT1-91/84 in the CFS patients
(32%) compared to the healthy controls (4%) is highly significant (p
< 0.01 by two-sided Fisher's Exact Test).
This suggests that a subpopulation of CFS patients may exist
who suffer from an abnormally low STAT1 response to interferons.
This immunodeficiency may underlie the increased
susceptibility to infections seen in many CFS patients.
These studies were supported by the
National CFIDS Foundation.
|
