Chronic Fatigue Syndrome (CFS), Persian Gulf War Illness (PGI), and fibromyalgia

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: BMC Neurol. 2005 Dec 1;5:22.

A chronic fatigue syndrome - related proteome in human cerebrospinal fluid.

Baraniuk JN, Casado B, Maibach H, Clauw DJ, Pannell LK, Hess S S.

town University Proteomics Laboratory, Division of Rheumatology,

Immunology & Allergy, Room B-105, Lower Level Kober-Cogan Building,

town University, 3800 Reservoir Road, N,W,, Washington DC 20007-2197,

USA. baraniuj@....

ABSTRACT : BACKGROUND : Chronic Fatigue Syndrome (CFS), Persian Gulf War

Illness (PGI), and fibromyalgia are overlapping symptom complexes without

objective markers or known pathophysiology. Neurological dysfunction is

common. We assessed cerebrospinal fluid to find proteins that were

differentially expressed in this CFS-spectrum of illnesses compared to

control subjects. METHODS : Cerebrospinal fluid specimens from 10 CFS, 10

PGI, and 10 control subjects (50 mul/subject) were pooled into one sample

per group (cohort 1). Cohort 2 of 12 control and 9 CFS subjects had their

fluids (200 mul/subject) assessed individually. After trypsin digestion,

peptides were analyzed by capillary chromatography,

quadrupole-time-of-flight mass spectrometry, peptide sequencing,

bioinformatic protein identification, and statistical analysis.

RESULTS : Pooled CFS and PGI samples shared 20 proteins that were not

detectable in the pooled control sample (cohort 1 CFS-related proteome).

Multilogistic regression analysis (GLM) of cohort 2 detected 10 proteins

that were shared by CFS individuals and the cohort 1 CFS-related proteome,

but were not detected in control samples. Detection of >/=1 of a select set

of 5 CFS-related proteins predicted CFS status with 80% concordance

(logistic model). The proteins were

alpha-1-macroglobulin,

amyloid precursor-like protein 1, keratin 16, orosomucoid 2 and

pigment epithelium-derived factor. Overall, 62 of 115 proteins were newly

described.

CONCLUSION : This pilot study detected an identical set of central nervous

system, innate immune and amyloidogenic proteins in cerebrospinal fluids

from two independent cohorts of subjects with overlapping CFS, PGI and

fibromyalgia. Although syndrome names and definitions were different, the

proteome and presumed pathological mechanism(s) may be shared.

PMID: 16321154 [PubMed - in process]

The regulation of pro-inflammatory gene expression induced by pigment

epithelium-derived factor in rat cultured microglial cells.

PMID: 15854760

Pigment epithelium-derived factor induces the production of chemokines by

rat microglia.

PMID: 15816038

Pigment epithelium-derived factor (PEDF) blocks angiotensin II signaling in

endothelial cells via suppression of NADPH oxidase: a novel anti-oxidative

mechanism of PEDF.

PMID: 15846509

a1-Microglobulin is one of the three original members of the lipocalin

superfamily. It has been found in mammals, birds, amphibians and fish and is

distributed in plasma and extravascular compartments of all organs.

a1-Microglobulin has a free cysteine side-chain located in a flexible loop,

giving the protein reductase and dehydrogenase properties with a broad

biological substrate specificity. Three lysyl residues located around the

opening of the lipocalin pocket carry yellow-brown modifications originating

from the binding and degradation of heme and kynurenin, the latter a

tryptophan metabolite. We have suggested that a1-microglobulin is involved

in defending tissues against oxidation by heme, kynurenin and reactive

oxygen species.

Recent reports suggest that a1m is involved in the defense against oxidative

tissue damage (oxidative stress). The proposed anti-oxidant mechanisms are

summarized in Figure 3. Pro-oxidants are constantly introduced to the human

body via the environment (air, food, etc) but are also produced endogenously

as metabolites in the normal homeostasis. 91 Increased amounts of

pro-oxidants are seen during inflammation, and oxidative stress is

considered to be a major factor in the development of many conditions such

as atherosclerosis, rheumatoid arthritis, ischemia/reperfusion injury, and

diabetes. The pro-oxidants undergo reactions forming reactive oxygen species

(ROS) and free radicals. These react with proteins, DNA and other molecules

of human tissues by oxidation. These oxidation reactions are often harmful

and may destroy the function of the target molecules (oxidative damage).

Heme, the prosthetic group of heme-proteins, is a prominent example of an

endogenous pro-oxidant and is harmful when released into the extracellular

environment.

Thus, a1m uses three major mechanisms to achieve anti-oxidation: (1)

scavenging heme and other pro-oxidants, (2) inhibiting oxidation reactions,

and (3) enzymatic reduction of harmful oxidation products. Additionally, a

fourth mechanism enhance its anti-oxidant action: heme- and

pro-oxidant-induced up-regulation of the synthesis of a1m 70 (see above).

Heme-Scavenging

Both a1m and its IgA-complex bind to the heme group. 34 - 36 The exposure of

a1m to erythrocyte membranes or purified hemoglobin leads to the binding of

heme and the formation of a truncated form of the protein (t-a1m) that lacks

the C-terminal tetrapeptide LIPR and has heme-degrading properties. A

pronounced yellow-brown colour was formed by incubating t-a1m with heme,

suggesting that at least some of the a1m-chromophores may be heme

degradation products. 34 The t-a1m form is present in urine and thus is

formed in vivo. 34 , 69 , 92 In chronic venous ulcers, an inflammatory

condition where free heme and iron released after hemolysis are considered

to be pathogenic factors, a1m was colocalized with heme and t-a1m was

continuously formed. 35 Based on these findings, a role as an extracellular

heme-scavenger was proposed for a1m. As mentioned above, the tryptophan

metabolite kynurenine is attached to lysyl residues in a1m from the urine of

hemodialysis patients. 32 Kynurenine metabolites are formed in tryptophan

metabolism and are pro-oxidants, i.e., may induce oxidative stress. 93 - 95

The binding of heme and kynurenin and the transformation of these substances

to chromophores may be two examples of a pro-oxidant scavenging mechanism of

a1m.

Inhibition of Oxidation and Enzymatic Reductase Activity

a1M inhibited the heme- and ROS-induced oxidation of collagen, low-density

lipoproteins (LDL), membrane lipids and whole cells. 96 a1M also removed

preformed oxidation products present on collagen and LDL. This suggests that

a1m may act as an oxidation repair factor. A possible mechanism for this may

be the enzymatic reductase/dehydrogenase properties recently described for

a1m. 97 Thus, the protein was capable of reducing heme proteins, free iron

and the synthetic compound nitroblue tetrazolium (NBT) using the electron

donors ascorbate and NADH/NADPH as cofactors. The thiol group of Cys34 and

the three lysyl residues of K92, K118 and K130 were found in the active

site, suggesting that the chromophore or chromophore formation is linked to

the reductase activities.

Immunoregulatory Properties

Due to its immunoregulatory properties, a1m is categorized as an

immunocalin. 98 It inhibits central events of the immune response in vitro.

Thus, the antigen-induced cell division of peripheral blood lymphocytes was

inhibited by a1m. 99 , 100 The effects were species independent, i.e.,

similar effects on human cells were obtained with human, rat, rabbit or

guinea pig a1m. 14 It was also shown that the antigen-induced interleukin-2

(IL-2) production by mouse T helper cell hybridomas was inhibited by human

a1m. 101 Furthermore, inflammatory responses of blood cells were inhibited

by a1m; these included migration 98 and chemotaxis 102 of neutrophil

granulocytes and the production of free radicals and IL-1b by peripheral

lymphocytes/monocytes. 88 Finally, at low serum concentrations, a strong

direct mitogenic effect by a1m on resting guinea pig and human lymphocytes

was seen. 100 , 103 , 104 At the high serum concentrations (10-20%) used to

measure the inhibition of antigen-stimulated lymphocytes, no direct

stimulation of the cell division was seen. It is possible that the

immunoregulatory effects of a1m are related to its anti-oxidant and

reductase activities, as ROS have been shown to be involved as (positive)

factors in cell signalling during lymphocyte activation (reviewed in refs.

105,106).

Cell Receptor

a1M has been shown to bind to the surface of various white blood cells,

including human peripheral B and T lymphocytes, human NK cells, 60 the human

histiocytic cell-line U937, 107 mouse peripheral B and T lymphocytes, 104

and mouse T helper cell hybridomas. 101 The binding is species independent,

specific for a1m, saturable and trypsin-sensitive, suggesting that a protein

that recognizes a conserved part of a1m is present on the surface of white

blood cells. The a1m-receptor on blood cells has not yet been identified,

however.

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