XMRV/CFS Inflammatory Signature -Lombardi et al

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Below you will find the abstract and the

Introduction-section of:

*Xenotropic Murine Leukemia Virus-related

Virus-associated Chronic Fatigue Syndrome

Reveals a Distinct Inflammatory Signature*

By Lombardi et al.

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in vivo 25: 307-314 (2011)

Xenotropic Murine Leukemia

Virus-related Virus-associated

Chronic Fatigue Syndrome

Reveals a Distinct Inflammatory

Signature

VINCENT C. LOMBARDI1, KATHRYN S. HAGEN1,

KENNETH W. HUNTER4, JOHN W. DIAMOND2†,

JULIE SMITH-GAGEN3, WEI YANG3 and JUDY A.

MIKOVITS1

1Whittemore Institute, University of

Nevada, Reno MS 0552, 1664 N. Virginia St., Reno,

NV 89557, U.S.A.;

2Triad Medical Center, 4600 Kietzke Lane M242,

Reno, NV 89502, U.S.A.;

3Nevada Center for Health Statistics and Informatics,

University of Nevada, 1664 N. Virginia St., Reno, NV

89557, U.S.A.;

4University of Nevada Reno, Department of

Microbiology and Immunology Applied Research

Facility,1664 N. Virginia St., MS 199, Reno, NV 89557

U.S.A.

Abstract.

Background:

The recent identification of xenotropic murine

leukemia virus-related virus (XMRV) in the blood of

patients with chronic fatigue syndrome (CFS)

establishes that a retrovirus may play a role in the

pathology in this disease.

Knowledge of the immune response might lead to a

better understanding of the role XMRV plays in this

syndrome.

Our objective was to investigate the cytokine and

chemokine response in XMRV-associated CFS.

Materials and Methods:

Using Luminex multi-analyte profiling technology, we

measured cytokine and chemokine values in the

plasma of XMRV-infected CFS patients and compared

these data to those of healthy controls.

Analysis was performed using the Gene Expression

Pattern Analysis Suite and the Random Forest tree

classification algorithm.

Results:

This study identifies a signature of 10 cytokines and

chemokines which correctly identifies XMRV/CFS

patients with 93% specificity and 96% sensitivity.

Conclusion:

These data show, for the first time, an

immunological pattern associated with XMRV/CFS.

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Introduction

Chronic fatigue syndrome (CFS) is a poorly

understood disease of unknown etiology, which is

commonly characterized by innate immune defects,

chronic immune activation and dysregulation, often

leading to neurological maladies [reviewed in (1)].

It can also involve other biological systems such as

the musculoskeletal, gastrointestinal and

endocrinological systems (2-4).

Although several common symptoms are primarily

reported and predominate, they may differ among

individuals, are often intermittent and can persist for

years, frequently resulting in substantial disability

(5).

Some of the most commonly reported physical

symptoms include muscle weakness and pain, tender

or swollen lymph nodes and chronic flu-like

symptoms (6).

Memory and concentration impairment, blurred

vision, dizziness and sleep abnormalities represent

some of the cognitive symptoms typically observed

while immunological symptoms often manifest

themselves through viral reactivation, RNase L

dysregulation, decreased natural killer (NK) cell

function and susceptibility to opportunistic infections

(7-12).

NK cell dysregulation may be associated with viral

reactivation or viral persistence and may also lead to

malignancy (13, 14).

Indeed, clinical observations corroborate pathological

manifestations in CFS as viral reactivations,

particularly herpes virus such as cytomegalovirus

(CMV), Epstein-Barr virus (EBV) and human herpes

virus-6 (HHV-6), are common occurrences (15-17).

Moreover, epidemiological studies have reported

increased incidences of lymphoma associated with

CFS outbreaks (18).

These clinical observations suggest that a

compromised innate immune system may play a role

in CFS pathology.

The completion of the human genome project

enabled positional cloning studies to identify the

RNASEL gene as the hereditary prostate cancer

allele-1 (HPC1) (19).

This discovery prompted Silverman and his

colleagues to search for a viral component to

hereditary prostate cancer.

Using a viral micro-array and tissue biopsies from

individuals with hereditary prostate cancer they

identified and sequenced the complete genome of a

novel human gammaretrovirus, very similar in

sequence to xenotropic murine leukemia virus and

therefore termed the new virus xenotropic murine

leukemia virus-related virus (XMRV) (20).

Subsequent studies performed in our laboratory

identified and isolated infectious XMRV in the blood

of 67% of CFS patients (21).

This work was performed using multiple techniques

including PCR, electron microscopy showing budding

viral particles, Western blot analysis of viral proteins

and serology confirming that infected patients

express antibodies to XMRV envelope proteins.

In addition, gene sequencing and phylogenetic

analysis confirmed these patients were indeed

infected with XMRV that was >99% identical to

previously published sequences but was obviously

distinct from the only existing XMRV molecular clone,

VP62 (20).

Taken together, this work clearly rules out any

possibility of gross contamination and additionally,

represents the first identification and isolation of

naturally occurring infectious XMRV.

The connection between CFS and XMRV was further

supported by the studies of Lo et al., who identified

murine leukemia virus (MLV)-related sequences in

the blood of 86% of CFS patients, further

establishing a retroviral association with CFS (22).

Presently, three families of retroviruses are known to

infect humans; the human immunodeficiency viruses

(HIV), the human T-cell leukemia viruses (HTLV) and

now the human murine leukemia-related viruses.

Both HIV and HTLV are known to dysregulate the

innate immune system and promote the production

of inflammatory cytokines and chemokines (23, 24).

In light of the association between XMRV and CFS, it

is not surprising that some of the most salient

observations in CFS are the differences in cytokines

and chemokines when compared to healthy controls

(8).

Previous reports, however, addressing the role of

these molecules in CFS have produced conflicting

results.

Much of this emerges from such hindrances as small

sample size, a limited number of cytokines surveyed

at one time, insufficient patient population

stratification, and insufficient negative control

subjects.

This has resulted in inconsistent reports in the

literature for a number of cytokines including

interleukins (IL) 6, 10 and 12.

In spite of these conflicting results, a number of

cytokines and chemokines have consistently been

show to be associated with different subgroups of

CFS.

For instance, Natelson et al. showed elevated levels

of IL-8 and IL-10 in the cerebral spinal fluid of

patients with sudden, influenza-like onset CFS when

compared to healthy controls (25).

Additionally, Chao et al. have show neopterin and

IL-6 to be up-regulated in subsets of CFS patients,

indicative of a pro-inflammatory immune condition

(26).

However, these studies did not analyze the complex

relationships between multiple cytokines and clinical

disease.

By applying conventional statistical analysis and

'machine logic' algorithms to the multiplex data, it is

possible to identify cytokines and chemokines that

are differentially expressed between two groups.

To support this premise, we have used the xMap®

multi-analyte profiling technology that allows

simultaneous measurements of multiple biomarkers

in serum or plasma.

In this study, a panel of 26 cytokines, chemokines

and growth and angiogenic factors were analyzed in

blood plasma of CFS patients and healthy control

subjects.

This study revealed a signature of 10 cytokines and

chemokines, which showed a specificity of 93% and

sensitivity of 96% in diagnosing XMRV-associated

CFS in this patient cohort.