Re: CFS Patent - Dr Gow et al

[Guest guest]

Interesting. Their protocol would basically treat inflammation, infection

(bacterial) and oxidative stress...which is what many protocols already address.

Good to know we're on the right track.

Thanks for posting.

Louella

---------------------------------

All-new - Fire up a more powerful email and get things done faster.

[Guest guest]

Has anyone had experience of using the drugs Dr Gow reccommends ?

Corticosteroid and minocycline ?

>

> Hi All,

> This has just been posted on a couple of lists. Looks very

interesting.

> BW,

> Sheila

>

> Source: European Patent Office

> Date: August 10, 2006

> URL: http://v3.espacenet.com/textdoc?

DB=EPODOC & IDX=WO2006082390 & F=0

> Ref: http://www.me-net.dds.nl/forward/genomics.html

>

>

> Bibliographic data of WO2006082390

> ----------------------------------

>

> Publication number: WO2006082390

> Publication date: 2006-08-10

> Inventor: GOW JOHN (GB);

> CHAUDHURI ABHIJIT (GB)

> Applicant: UNIV GLASGOW (GB);

> GOW JOHN (GB);

> CHAUDHURI ABHIJIT (GB)

> Classification: international: C12Q1/68; G01N33/53; C12Q1/68;

> G01N33/53;

> european: Application number: WO2006GB00332

20060201

> Priority number(s): GB20050002042 20050201

> View INPADOC patent family

> Cited documents: XP002361324

> XP002377235

> XP002377236

> XP005062766

> XP002377237

>

>

> Abstract of WO2006082390

> ------------------------

>

> The invention relates to materials and methods for diagnosis and

> treatment of

> chronic fatigue syndrome/myalgic encephalitis. A number of genes

are

> identified

> which are expressed at abnormal levels in patients affected by

CFS/ME as

> compared to normal healthy individuals. These genes include those

> encoding

> defensin a1, haemoglobin, CXCR4, tubulin beta 1, serine/threonine

kinase

> 17B,

> HLA DRss4 and prostaglandin D2 synthase. The genes identified

provide

> objective

> disease markers that may be used in diagnostic tests to support the

> diagnosis of

> CFS/ME or for monitoring the effectiveness of therapy. They also

provide

> a

> rational basis for classifying CFS/ME patients according to the

> biochemical

> lesion underlying their symptoms and enable provision of

appropriate

> targeted

> therapies.

>

>

> Description of WO2006082390

> ---------------------------

>

> [0001] Materials and Methods for Diagnosis and Treatment of Chronic

> Fatigue

>

> [0002] Syndrome

>

> [0003] Field of the Invention The invention relates to chronic

fatigue

> syndrome/myalgic encephalitis, and in particular to materials and

methods

> for

> its diagnosis and treatment.

>

> [0004] Background to the Invention Chronic fatigue syndrome (CFS),

also

> known

> as Myalgic

>

> [0005] Encephalomyelitis (ME), is an acquired disorder with long

term

> disability<1>. The illness affects both children and adults and is

> characterised

> by persistent or relapsing fatigue of sufficient severity to

interfere

> with

> normal function. In addition, patients experience impairment in

short

> term

> memory and concentration, muscle pain and prolonged post-exertional

> malaise<2>.

> The estimated overall prevalence of CFS/ME in the community is

> approximately

> 0.2-0.4% in adults and 0.07% in adolescents and children<3>.

>

> [0006] The pathogenesis of CFS/ME is unknown. Diagnosis of CFS/ME

> according to

> the internationally accepted definition (modified CDC criteria) is

> essentially

> based on exclusion of known medical and psychiatric diseases<2>.

As yet,

> there

> is no specific or sensitive diagnostic test that positively

establishes

> and/or

> supports the clinical diagnosis of CFS/ME.

>

> [0007] Nevertheless, in 2002, following the publication of a UK

> Government

> Working Party report, Professor Sir Liam son confirmed that

CFS/ME

> is a

> debilitating and distressing condition affecting many people which

should

> be

> classed alongside other diseases such as multiple sclerosis and

motor

> neurone

> disease " .

>

> [0008] A significant proportion of patients report antecedent

history of

> community acquired viral or bacterial infections <4>'<5>'<6>. in

> addition, it is

> clear that patients with CFS/ME have a hypoactive

> hypothalamic-pituitary-adrenal

> (HPA) axis with altered neuroendocrine regulation affecting

> neurotransmitters

> such as monoamines (norepinephrine, serotonin and dopamine) and

> acetylcholine in

> the central nervous system<4>. Recent studies have demonstrated

that

> viruses can

> affect neurotransmitter functions<7>'<8>.

>

> [0009] At the cellular level, fatigue has been linked with

alterations in

> the

> cell membrane ion-channel traffic and ATPase system<9>. ATPases

are also

> linked

> with neurotransmitter release (e. g. dopamine) <10> and cellular

energy

> metabolism via creatine phosphatase. Increased

>

> [0010] ATPase activity has been reported in muscle biopsies from

patients

> with

> CFS <11>' <12>. Previous work<11>' <13> has raised the possibility

that

> patients

> with CFS may have an ion channel dysfunction. This dysfunction

might be

> induced

> by changes in the ion channel function, neurotransmitters involved

in

> " gating "

> the channel or by a shift in the balance of the cellular " energy

charge " ,

> i.e. the ratio between ATP and ADP that is normally a function of

the

> ATPase

> activity.

>

> [0011] One previous study<43> has identified a number of genes

> differentially

> expressed between PBMCs from CFS/ME patients and healthy controls.

A

> number of

> the genes identified are implicated in various aspects of

immunological

> function, from which the authors concluded that some kind of

> immunological

> dysfunction may be involved in pathogenesis of CFS/ME. The authors

were

> unable

> to relate the observed expression patterns to any functional model

of

> disease

> etiology or pathology, and did not suggest that any of the genes

which

> they

> identified could serve as useful biomarkers for CFS/ME. Their

conclusions

> are

> consistent with previous studies which have suggested immunological

> abnormality

> in CFS/ME (Refs. 44 to 48) but the precise mechanism of

immunological

> dysfunction in CFS/ME has not been established.

>

> [0012] Summary of the Invention

>

> [0013] The present inventors have identified a number of genes

which are

> expressed at abnormal levels in patients affected by CFS/ME as

compared

> to

> normal healthy individuals. In contrast to the earlier studies

described

> above,

> the present inventors have been able to use the expression

patterns of

> these

> genes to establish functional models of various aspects of the

pathology

> of

> CFS/ME, which explain many of the symptoms observed in affected

> individuals.

> These provide a rational basis for classifying CFS/ME patients

according

> to the

> biochemical lesion underlying their symptoms and enable appropriate

> targeted

> therapies to be provided for the first time.

>

> [0014] The genes identified in the present invention provide obj

ective

> disease

> markers that may be used in diagnostic tests to support the

diagnosis of

> CFS/ME

> or in other applications. For example, the tests may enable

deselection

> of

> inappropriately diagnosed patients who have an alternative

diagnosis for

> their

> fatigue symptoms. The tests may also be used to classify patients

> according to

> the particular biochemical basis for their symptoms. In turn, this

may

> enable a

> clinician to identify therapies which would be appropriate and

rule out

> those

> which would not. The tests may also be applied as an outcome

measure for

> interventional trials in CFS/ME, not least because no specific

treatment

> is

> currently considered to be effective in the patient population

defined by

> the

> clinical criteria. Further, the tests may offer support to CFS/ME

> patients for

> their claims for disability and insurance. Finally, depending on

the

> specificity and sensitivity of the test, one may apply it to screen

> patients

> with symptoms of chronic fatigue to identify or exclude CFS/ME.

Given the

> fact

> that chronic fatigue as a symptom is ten times commoner in the

population

> (2%)

> than CFS/ME, an estimated size of the market for a screening test

is at

> least

> 10 million in the UK alone.

>

> [0015] Thus the present invention provides a method for

investigating

> whether a

> test subj ect is affected by chronic fatigue syndrome/myalgic

> encephalomyelitis

> (CFS/ME), the method comprising providing a biological sample from

the

> subj ect

> and determining the level in the sample of a biomarker for CFS/ME,

> wherein the

> biomarker is an expression product of a gene shown in Table 1.

Genes in

> Table 1

> have been found to be overexpressed in CFS/ME compared to

unaffected

> individuals.

>

> [0016] The method may comprise determining the level, in the

sample, of a

> plurality of biomarkers (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10,

15, 20

> or more biomarkers), wherein each of the biomarkers is an

expression

> product

> of a gene shown in Table 1.

>

> [0017] The method may comprise the step of determining the level

in the

> sample

> of one or more further biomarkers, being expression products of

one or

> more

> genes from Table 6. Indeed, an expression product from any one of

the

> genes of

> Table 6 may be used as a biomarker for CFS/ME, either alone or in

> combination

> with other genes of Table 6 or of Tables 1 to 5.

>

> [0018] The method may be considered to provide an expression

profile of

> the one

> or more biomarkers for CFS/ME, for the test subj ect, at the time

of

> sampling.

> By " expression profile " is meant a set of data relating to the

level of

> expression of one or more of the relevant biomarkers in a test

subject,

> in a

> form which allows comparison with comparable expression profiles

(e.g.

> from

> affected and/or unaffected individuals), in order to assist in the

> determination of whether or not the subject is affected by CFS/ME.

>

> [0019] The method typically involves correlating the results

obtained

> with a

> probability that the subj ect is affected by CFS/ME. In order to

assist

> with

> this correlation, the method of the invention may comprise the

step of

> comparing

> the expression profile for the test subj ect with one or

more " reference "

> expression profiles, that is to say one or more expression

profiles

> characteristic of unaffected subj ects (i.e. subj ects not

suffering from

> CFS/ME), and/or one or more expression profiles characteristic of

> affected subj

> ects (i.e. subj ects not suffering from CFS/ME). Thus the level of

> expression of one or more of the said biomarkers in the test subj

ect is

> typically compared with that characteristic of affected and/or

unaffected

> individuals.

>

> [0020] The reference expression profiles may be profiles previously

> derived from

> healthy or affected individuals, or may be artificial profiles

which

> display

> expression levels of the relevant biomarkers which are

characteristic of

> the

> relevant group. For example, they may be computer-generated from a

> plurality of

> profiles previously derived from appropriate individuals. The

profile (s)

> characteristic of affected subj ects may be divided into

subgroups, e.g.

> according to their symptoms, as described in more detail below.

>

> [0021] Taken alone, the expression profile for the test subj ect

may not

> provide

> an absolute diagnosis of CFS/ME. Normally, a clinician will also

take

> account

> of the physical and/or psychological symptoms of the subj ect in

order to

> reach

> a diagnosis. However the expression profile generated by the

methods of

> the

> invention provides useful data to help the clinician confirm or

rej ect a

> preliminary diagnosis based on physical and psychological symptoms

alone.

> A

> finding that one or more of these genes is upregulated in a

particular

> individual may therefore provide support for a diagnosis of CFS/ME.

>

> [0022] By " upregulated " or " overexpressed " is meant that the gene

in

> question

> shows at least a two fold increase in expression at the level of

> r[alpha]RNA

> and/or protein as compared to the level observed in unaffected

> (preferably

> healthy) individuals. If desired, the level of overexpression

required to

> regard a result as positive may be set higher than this, e.g. a

three,

> four,

> five, six, seven, eight, nine or ten fold increase as compared to

> unaffected

> individuals, or higher if required.

>

> [0023] The genes in Table 1 not only provide biomarkers for

CFS/ME, they

> also

> provide the first evidence of specific biochemical pathways which

may be

> dysregulated in CFS/ME. They can be clustered into a number of

subgroups,

> based on their involvement in the same or related biochemical

pathways

> whose

> dysregulation is likely to play a role in either the underlying

cause or

> the

> symptoms of the disease state. Previously it has not been possible

to

> identify

> such pathways, which is a primary reason why no generally-

recognised

> therapies

> exist for CFS/ME. The subgroups of genes identified by the present

> inventors

> provide an explanation for many of the symptoms displayed by those

> affected by

> CFS/ME, suggest rational therapies for the condition, and provide

means

> for

> monitoring the efficacy of any therapy administered.

>

> [0024] Without wishing to be bound by any particular theory, the

> inventors

> believe that CFS/ME is not a genetic disease caused by a single or

> multiple gene

> defects. Rather, they believe CFS/ME to be an acquired condition

where

> there is

> a shift in the functional systems of a select number of genes

regulating

> specific biological functions (e.g. infection and immunity, cell

membrane

> function and cell cycle) Based on the expression patterns they have

> observed,

> they have established a model of " hub " and " network " genes which

> emphasises the

> interrelation of these key genes, with the hub genes being the

control

> centre

> and the network genes being largely the effector arm of this

functional

> system.

> This set of hub and network genes defines the functional shift in

the

> biological

> systems of patients who continue to have symptoms due to CFS/ME.

>

> [0025] Thus the genes of Table 1 are subdivided into " hub " genes

and

>

> [0026] " network " genes. Essentially, in any particular pathway the

hub

> gene(s)

> can be considered to lie upstream of the network genes, in that

> dysregulation

> of the hub gene is likely to lead (directly or indirectly) to

> dysregulation of

> the downstream genes. In Table 1, the hub genes are shown in

section A,

> and the

> network genes in section B.

>

> [0027] Certain genes which appear to be highly significant (e.g.

based on

> the

> difference in their expression between the disease state and the

normal

> state)

> have been allocated to the " hub " group even where no related set of

> downstream

> " network " genes is specifically identified.

>

> [0028] Exemplary groups of genes identified in Table 1 are

detailed below.

>

> [0029] Defensin [alpha]1, CXCR4 and lactotransferrin (LTF) are all

> involved in

> the response to intracellular infection. The network genes

associated

> with

> these hub genes include defensin [alpha]4, integrin [alpha]2B,

integrin

> [beta]3,

> arginase 1, arginase 2, thrombospondin 1, membrane associated

protein 17

> (MAP 17), Charcot Leyden Crystal Protein (CLC) and chondroitin

sulphate

> proteoglycan 2 (versican). These genes are shown in Table 2.

>

> [0030] Haemoglobin Y (foetal haemoglobin) is part of the oxidative

stress

> response pathway, and is only expressed under conditions of

oxidative

> stress.

> Network genes associated with the oxidative stress pathway include

other

> haemoglobin genes including haemoglobin alpha 1,

> prostaglandin-endoperoxide

> synthase 1 and prostaglandin- endoperoxide synthase 2. These genes

are

> shown in

> Table 3. However it will be noted that Table 6 includes genes for

> haemoglobins

> alpha, beta, gamma and delta. Any one of the haemoglobin genes of

Table 6

> may

> be used as a biomarker for CFS/ME, and may be used as well as, or

in

> place of

> haemoglobin gamma or alpha if desired. For simplicity, though,

reference

> will be

> made primarily to haemoglobin gamma.

>

> [0031] Serine/threonine kinase 17B (STK17B) is implicated in

apoptosis,

> as are

> the network genes caspase 1, dynamin 1-like, and phosphatidyl

serine

> binding

> protein. These genes are shown in Table 4.

>

> [0032] The MHC class II gene HLA-DR[beta]4 is upregulated in CFS/ME

> implying a

> shift from antigen presentation by MHC class I to MHC II

presentation.

> Associated network genes include HLA-DQ[beta]1 and the

immunoglobulin

> heavy

> chain [gamma]3 (IgG3). These genes are shown in Table 5. IgG1 may

be used

> as a

> bior[alpha]arker as well as, or in place of, IgG3.

>

> [0033] Methods for investigating whether a test subj ect is

affected by

> CFS/ME

> may therefore comprise determining the level of at least one gene

from

> each of

> at least two of Tables 2, 3, 4 and 5, and preferably from each of

three

> or

> four of said Tables. In preferred embodiments, at least one gene

from

> each

> said Table is a hub gene.

>

> [0034] Preferred genes of Table 1 are defensin [alpha]1,

haemoglobin

> [gamma],

> CXCR4, tubulin beta 1 and HLA DR[beta]4. The method may involve

testing

> expression of any one, two, three, four or all five of these

genes, and

> optionally further hub genes of Table 1A.

>

> [0035] Additionally or alternatively, the method may involve

testing

> expression

> of one or more of TSP 1, caspase 1 and/or IgG3.

>

> [0036] The method may also comprise the step of determining the

level of

> an

> expression product of prostaglandin D2 synthase in sample. In

contrast to

> the

> genes of Table 1, prostaglandin D2 synthase is found to be

downregulated

> in

> individuals suffering from CFS/ME as compared to normal controls.

Thus a

> finding that prostaglandin D2 synthase is downregulated in a

particular

> individual may provide support for a diagnosis of CFS/ME.

>

> [0037] By " downregulated " or " underexpressed " is meant that

prostaglandin

> D2

> synthase shows at least two fold higher expression at the level of

mRNA

> and/or

> protein in unaffected (preferably healthy) individuals than is

observed

> in the

> test subj ect. If desired, the level of downregulation required to

regard

> a

> result as positive may require the level found in unaffected

individuals

> to be

> three, four, five, six, seven, eight, nine or ten fold higher than

in

> affected

> individuals. The method may further involve the step of

determining the

> level,

> in a biological sample from the subj ect, of the peptide QYNAD, as

> described in

> more detail below. The level of the peptide in the sample may form

part

> of the

> expression profile established for the subj ect. An elevated level

of the

> peptide as compared to unaffected individuals may be indicative of

> CFS/ME. By

> an " elevated " level is meant at least twice the level found in

unaffected

> healthy controls, and preferably at least 5 times or at least 10

time the

> level

> found in unaffected healthy controls.

>

> [0038] It will be appreciated that individual patients who each

satisfy

> the

> criteria for CFS/ME may nevertheless present a range of very

different

> symptoms.

> Some of these can be explained by the groups of genes identified

herein.

>

> [0039] For example, CFS/ME has previously been suggested to involve

> immune

> dysfunction. Some CFS/ME sufferers are particularly prone to

infection by

> viruses (e.g. influenza) and other pathogens; indeed some suffer

> recurrent

> infections. Others are affected by atopic/allergic symptoms. These

> symptoms

> may be associated with the apparent shift from Type I to Type II

antigen

> presentation by the MHC, which could impair the body' s ability to

deal

> efficiently with infections and could also exacerbate

allergy/atopy.

>

> [0040] Some CFS/ME sufferers describe their body as feeling

prematurely

> aged,

> and can display restricted mobility characteristic of much older

> individuals.

> This might be explained by excessive apoptosis, particularly in the

> central

> nervous system. Therefore patients having these symptoms might be

> expected to

> show increased expression of serine/threonine kinase 17B (STK17B)

and its

> associated network genes.

>

> [0041] Oxidative stress is also a cause of apoptosis, particularly

in the

> nervous system. Thus patients with increased expression of

haemoglobin Y

> and its

> associated network genes may also show, or be at risk of,

accelerated

> programmed

> cell death (increased level of apoptosis), particularly neuronal

> apoptosis.

> Features of oxidative stress and neuronal apoptosis may include

ageing,

> cognitive impairment and chronic pain.

>

> [0042] The genes of Table 2 are implicated in cellular protection

> ( " defence " )

> against viral or bacterial infections. Thus an overexpressed

defensin

> gene in

> CFS/ME would be consistent with immune activation and correlate

with

> symptoms of

> recurrent influenza-type symptoms, sore throat and lymph node

enlargement

> experienced by patients with CFS/ME.

>

> [0043] In a further aspect, the invention therefore provides a

method for

> classifying a subj ect affected by CFS/ME, the method comprising

> providing a

> biological sample from the subject and determining the level in the

> sample of a

> biomarker for CFS/ME, wherein the biomarker is an expression

product of a

> gene

> shown in one or more of Tables 2 to 5.

>

> [0044] Preferably the biomarker is a hub gene from one of Tables

2, 3, 4

> or 5.

> More preferably, the method comprises determining the expression

level of

> hub

> genes from each of two, three or all four of Tables 2, 3, 4 or 5.

The

> method

> may also comprise determining network genes from one, two, three

or all

> four of

> Tables 2, 3, 4 and 5.

>

> [0045] The individual can then be assigned to a subgroup of CFS/ME,

> dependent on

> which group or groups of genes are found to be upregulated. Thus,

for

> example,

> the subj ect may be classified as being affected by one or more of

> oxidative

> stress, excessive apoptosis, and immunological dysregulation (MHC

I to II

> shift). The assignment step may involve comparing the expression

profile

> obtained from the subj ect with one or more expression profiles

> characteristic

> of individuals previously assigned to one or more subgroups of

CFS/ME. It

> will

> be appreciated that the classification may be performed using the

same

> expression profile as that established for determining whether the

subj

> ect is

> affected by CFS/ME.

>

> [0046] Individuals in which these particular pathways are

dysregulated

> may be

> suitable for treatment by the methods described below. Therefore

in a

> further

> aspect the invention provides a method of determining whether an

> individual

> affected by CFS/ME is suitable for treatment using such a therapy.

The

> method

> comprises determining whether the individual is affected by one or

more

> of

> oxidative stress, excessive apoptosis, and immunological

dysregulation as

> described above, and optionally prescribing a suitable treatment

> depending on

> the outcome. These treatments are described in detail below.

>

> [0047] Expression of individual biomarkers for CFS/ME (genes from

Tables 1

> to 5) may differ slightly between independent samples, leading to

> slightly

> different expression profiles for individual samples. However,

these

> particular

> genes may provide a characteristic pattern of expression

(expression

> profile) in

> an affected individual (i.e. one suffering from CFS/ME) that is

> recognisably

> different from that in an unaffected individual (i.e. one not

suffering

> from

> CFS/ME).

>

> [0048] By creating a number of expression profiles from a number

of known

> affected and unaffected samples, it is possible to create a

library of

> profiles

> for both sample types. The greater the number of expression

profiles, the

> easier it is to create a reliable characteristic expression profile

> standard

> (i.e. including statistical variation) that can be used as a

control in a

> diagnostic assay. Thus, a standard profile may be one that is

devised

> from a

> plurality of individual expression profiles and devised within

> statistical

> variation to represent either the affected or unaffected profile.

The

> determination of the expression profile may be computerised and

may be

> carried

> out within certain previously set parameters, to avoid false

positives

> and false

> negatives.

>

> [0049] The computer may then be able to provide an expression

profile

> standard

> characteristic of an affected sample and a normal sample. The

determined

> expression profiles may then be used to classify test samples as

affected

> or

> unaffected as a way of diagnosis.

>

> [0050] Thus, in a further aspect the invention provides a method of

> creating a

> library of expression profiles for use in determining whether an

> individual is

> affected by CFS/ME, the method comprising

>

> [0051] (a) providing biological samples from a plurality of

individuals

> affected

> by CFS/ME, and determining the level in each sample of one or more

> biomarkers

> for CFS/ME to create a plurality of expression profiles from

affected

> individuals;

>

> [0052] ( providing biological samples from a plurality of

individuals

> not

> affected by CFS/ME, and determining the level in the sample of

said one

> or more

> biomarkers for CFS/ME to create a plurality of expression profiles

from

> unaffected individuals;

>

> [0053] wherein the biomarkers comprise expression products of one

or more

> genes

> shown in Table 1.

>

> [0054] Typically, the method comprises the step of retrievably

storing

> each of

> the expression profiles on a computer data carrier, in order to

create a

> database of expression profiles for both affected and unaffected

> individuals.

>

> [0055] The invention further provides an expression profile

database,

> comprising

> a plurality of expression profiles of biomarkers for CFS/ME from

affected

> and

> unaffected individuals, wherein the biomarkers comprise expression

> products of

> one or more genes shown in Table 1.

>

> [0056] It will be appreciated that the profiles may be classified

> according to

> their expression levels of the various groups of genes shown in

Tables 2

> to 5 as

> already described. The combinations of biomarkers analysed in

> establishing the

> expression profiles may therefore be chosen as described above.

>

> [0057] The expression profiles may comprise data relating to the

level,

> in

> biological samples from the subj ects, of the peptide QYNAD. If

required,

> the

> methods may therefore comprise the step of determining the level

of this

> peptide

> in biological samples from the subjects.

>

> [0058] At present there are no generally acknowledged treatments

for

> CFS/ME.

> One problem which has faced researchers attempting to develop and

> evaluate

> suitable therapies is the lack of objective testable criteria to

> determine their

> efficacy. The present invention provides a useful tool for

monitoring the

> efficacy of an interventional treatment for CFS/ME, by studying the

> effect of

> the therapy, over time, on an individual' s expression profile of

the

> biomarkers

> described in Tables 1 to 5. The methods described may therefore be

used

> to

> evaluate the effectiveness of a test treatment, e.g. by testing it

on a

> population of subjects affected by CFS/ME, or to investigate an

> individual' s

> response to a particular therapy, to see whether or not they are

> responding

> appropriately.

>

> [0059] Thus in a further aspect the invention provides a method of

> determining

> the efficacy of a treatment for CFS/ME comprising the steps of:

>

> [0060] (a) providing a biological sample from a subj ect affected

by

> CFS/ME who

> has been subj ected to said treatment, ( determining the level

in said

> sample

> of one or more biomarkers for CFS/ME to create an expression

profile for

> said

> subj ect, and

>

> [0061] © comparing said expression profile with

>

> [0062] i) a comparable expression profile obtained from said test

subj

> ect

> before initiation of said treatment, and/or

>

> [0063] ii) a comparable expression profile obtained from said test

subj

> ect at

> an earlier stage of said treatment, and/or

>

> [0064] iii) a comparable expression profile characteristic of a

subject

> who is

> unaffected by CFS/ME,

>

> [0065] wherein the one or more biomarkers for CFS/ME are expression

> products of

> one or more genes shown in Tables 1 to 5. Preferred combinations of

> biomarkers

> have already been described in relation to earlier aspects of the

> invention.

>

> [0066] In general, a treatment may be considered to be effective

if the

> subj

> ect' s expression profile after treatment shows that the

expression level

> of one

> or more of the biomarkers is reduced compared to its level before

> treatment, or

> its level earlier in a course of the treatment. Preferably the

subj ect'

> s

> expression profile after treatment approaches a profile

characteristic of

> an

> individual unaffected by CFS/ME. That is to say, the expression

level of

> each

> of the biomarkers falls within the normal range found in unaffected

> individuals.

>

> [0067] Some treatments may be targeted to particular pathways

known to be

> dysregulated in CFS/ME. In such cases, it may be desirable to

follow the

> effect of the treatment on the expression levels of genes in the

pathway

> likely

> to be modulated by that treatment. For example, a treatment which

is

> intended

> to reduce oxidative stress, apoptosis or immune dysfunction

> (characterised by

> MHC shift) may have a particular effect on the expression levels

of the

> genes in

> Tables 3, 4 and 5 respectively. The biomarkers chosen to study the

> effects of

> the treatment may be chosen accordingly. Preferred combinations of

> biomarkers

> from these Tables have already been described.

>

> [0068] The method may further comprise determining the effect of

the

> treatment

> on the level of the peptide QYNAD in a biological sample from the

test

> subject.

>

> [0069] In the diagnostic and analytical methods described herein,

the

> expression

> levels of the chosen biomarker (s) for CFS/ME are preferably

determined

> using

> peripheral blood mononuclear cells (PBMCs) from the test subj ect.

> Therefore

> the biological sample used to establish the expression profile is

> preferably a

> blood sample or cells isolated from a blood sample. If desired, the

> method may

> comprise the steps of enriching PBMCs in the sample, or isolating

PBMCs

> from the

> sample.

>

> [0070] It will be understood that the methods described are

generally

> performed

> on a biological sample which has been isolated from the test

subject. The

> method may, but need not, comprise the actual step of isolating the

> sample from

> the test subj ect, e.g. by taking a blood sample.

>

> [0071] Typically, the expression level of each of the biomarkers is

> determined

> by contacting the sample with a binding agent capable of binding

> specifically to

> an expression product of the genes encoding that biomarker (a gene

of

> Table 1, 2,

> 3, 4, 5 or 6). Binding between the agent and an expression product

> present

> in the sample is then determined. If it is desired to determine the

> expression

> level of more than one gene, then the sample may be contacted with

a

> plurality

> of binding agents, either simultaneously or sequentially, each

binding

> agent

> being capable of binding specifically and individually to an

expression

> product

> of one of the biomarker genes. Binding agents capable of binding to

> nucleic

> acid expression products (e.g. mRNA, pre-mRNA) are typically

nucleic acid

> primers or probes having a sequence of, or complementary to, a

portion of

> the

> nucleic acid expression product. This enables the binding agent to

> hybridise

> under suitable conditions with the nucleic acid expression product

> itself, e.g.

> in a Northern blot or in situ hybridisation assay, or to a cDNA

copy of

> the

> nucleic acid expression product, e.g. in a RT-PCR assay, Southern

blot or

> microarray assay.

>

> [0072] Binding agents capable of binding to polypeptide expression

> products

> include ligands and receptors for the polypeptide in question.

> Particularly

> preferred examples of binding agents are antibodies specific for

(e.g.

> raised

> against) the desired polypeptide, or fragments thereof comprising

an

> antigen

> binding site. These may be used in a variety of immunological assay

> methods,

> including Western blots and ELISA assays, as well as in microarray

assays.

>

> [0073] It may be desirable to isolate expression products or

particular

> fractions thereof (e.g. total RNA, mRNA, total protein, soluble

proteins

> or

> membrane proteins) from the sample before contacting them with the

> binding agent.

> The extent to which this is necessary will vary depending on the

chosen

> assay

> method.

>

> [0074] Simple immunological assays (such as ELISA assays) or PCR-

based

> assays

> using ten biomarkers or less (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10

> biomarkers) are particularly preferred, because they can readily be

> adapted to

> analyse large numbers of samples in a relatively short space of

time, at

> relatively low cost. Such assays are also well- suited to

automation.

> Preferred

> markers and combinations of markers are as described elsewhere in

this

> specification.

>

> [0075] The diagnostic and analytical methods described above may

also

> comprise

> determining the level, in a biological sample from the subject, of

the

> peptide

> QYNAD. It is believed that the peptide may be found in serum and

> cerebrospinal

> fluid (CSF) in most or all of the population, but it is found at

elevated

> levels

> in subjects suffering from inflammatory and immunological

disorders of

> the

> nervous system (such as multiple sclerosis and Guillain-Barre

syndrome).

> The

> present inventors have now established that it is also found at

> significantly

> elevated levels in subj ects suffering from CFS/ME.

>

> [0076] The biological sample used for determining the peptide

level may

> be a

> blood sample (or serum derived therefrom). Thus, conveniently, the

same

> blood

> sample may be used for determining levels of the peptide and the

other

> biomarkers of CFS/ME. Alternatively, a different sample may be

used, in

> particular, a sample of cerebrospinal fluid (CSF) where available.

>

> [0077] The level of the peptide may be determined as described by

> Brinkmeier et

> al.<16>, e.g. by gel filtration chromatography. Alternatively

> conventional

> immunoassays such as ELISAs or Western blots may be used, employing

> antibodies

> raised against the peptide.

>

> [0078] The assay for the peptide is generally performed on a sample

> isolated

> from the test subj ect. The method of the invention need not

comprise the

> step

> of actually isolating the sample from the test subj ect.

>

> [0079] In a further aspect, the invention provides a kit for use

in a

> diagnostic

> or analytical method as described herein, the kit comprising a

plurality

> of

> binding agents, each capable of binding specifically and

individually to

> an

> expression product of one of the genes of Table I1 or the peptide

QYNAD.

> Thus

> the kit includes binding agents specific for expression products

of two

> or more

> genes of Tables 1, or at least one of the genes of Table 1 and the

> peptide QYNAD.

> The binding agents provided in the kit may be capable of binding

> specifically

> and individually to expression products of 2, 3, 4, 5, 6, 7, 8, 9,

10,

> 15,

> 20 or more of the genes of Table 1 and optionally the peptide

QYNAD.

>

> [0080] The kit is suitable for use in the methods of the invention

> described in

> this specification, and may comprise instructions for performing

one or

> more

> methods of the invention.

>

> [0081] The binding agents may be immobilised on one or more solid

> supports.

> Discrete supports may each carry only one type of binding agent.

For

> example,

> distinct populations of beads may each carry one type of binding

agent.

> Alternatively, a single support may carry more than one type of

binding

> agent.

> Indeed, one support (e.g. a microarray chip) may carry all of the

> different

> types of binding agent provided with the kit.

>

> [0082] In addition, the kit may comprise one or more binding agents

> capable of

> binding specifically to an expression product of a control gene

which is

> not

> differentially expressed between individuals affected and

unaffected by

> CFS/ME.

> The level of expression from this control gene may be measured m

order to

> assist

> in quantification of the expression products of the genes of Table

1,

> and/or

> for quality assurance of an assay performed using the kit.

Preferably a

> control

> gene is chosen which is constitutively expressed in the cells of

the

> biological

> sample (i.e. always expressed, at substantially the same level,

under

> substantially all conditions). Such genes are often referred to as

> " housekeeping " genes. Examples include glyceraldehyde phosphate

> dehydrogenase

> (GAPDH), [beta]-actin, and abl (ableson tyrosine kinase).

>

> [0083] The kit may comprise yet further binding agents capable of

binding

> to

> expression products of other biomarker genes or control genes.

However, m

> preferred embodiments, the kit comprises binding agents for

expression

> products

> of less than 1000 different genes, e.g. less than 500 different

genes,

> less

> than 100, less than 50, less than 40, less than 30, less than 20,

or less

> than 10 different genes. As explained above, the groups of genes

> identified by

> the present inventors also suggest specific therapies for CFS/ME.

>

> [0084] Patients displaying signs of oxidative stress may be

treated with

> an

> anti-oxidant. Thus the invention provides a method of treating

CFS/ME in

> an

> individual suffering therefrom, comprising administering an

effective

> amount of

> an anti-oxidant.

>

> [0085] Therapeutically effective amounts of a corticosteroid and/or

> minocyline

> may also be administered to the subject in conjunction with the

> anti-oxidant.

> As will be appreciated, the individual active agents may be

administered

> individually (in two or more separate compositions) or together

(in the

> same

> composition).

>

> [0086] The invention further provides the use of an anti-oxidant

in the

> preparation of a medicament for the treatment of CFS/ME. The

medicament

> may be

> formulated for administration in conjunction with a corticosteroid

and/or

> minocycline. Alternatively, the medicament may comprise a

corticosteroid

> and/or

> minocycline.

>

> [0087] Examples of suitable anti-oxidants include coenzyme Q10 and

> inhibitors of

> cyclooxygenase (COX) enzymes, particularly COX II enzymes, such as

> celecoxib (

> 4- [5- (4-methyl[rho]henyl) -3- (trifluoromethyl) - 1H-pyrazol-1-

yl]

> benzenesulfonamide).

>

> [0088] Patients showing evidence of abnormal apoptosis may be

treated

> with

> minocyline, which is an inhibitor of caspase 1, shown here to be

> upregulated in

> individuals affected by CFS/ME. Minocyline has previously been

suggested

> for

> treatment of various neurological disorders including stroke,

multiple

> sclerosis,

> spinal cord injury, Parkinson' s disease, Huntington' s disease

and

> amylotrophic lateral sclerosis - see Wee Young et al., Lancet

Neurology,

> 2004,

> 744-751 for review. Thus the invention provides a method of

treating

> CFS/ME

> in an individual suffering therefrom, comprising administering an

> effective

> amount of minocycline.

>

> [0089] Therapeutically effective amounts of a corticosteroid

and/or an

> anti-oxidant may also be administered to the subject in

conjunction with

> the

> minocycline. As will be appreciated, the individual active agents

may be

> administered individually (in two or more separate compositions) or

> together

> (in the same composition).

>

> [0090] The invention further provides the use of minocycline in the

> preparation

> of a medicament for the treatment of CFS/ME. The medicament may be

> formulated

> for administration in conjunction with a corticosteroid and/or an

> anti-oxidant.

> Alternatively, the medicament may comprise a corticosteroid and/or

an

> anti-oxidant.

>

> [0091] Oxidative stress often gives rise to apoptosis. Therefore

it may

> be

> advisable to treat patients showing signs of oxidative stress with

an

> apoptosis

> treatment such as minocycline as a precautionary measure if they

do not

> already

> show upregulation of genes involved in apoptosis.

>

> [0092] Patients showing signs of immune dysfunction, and

particularly the

> Type I

> to II shift of MHC expression, may benefit from treatment with a

> corticosteroid.

> This would address the hypocortisolism previously reported in some

CFS/ME

> patients.

>

> [0093] Thus the invention provides a method of treating CFS/ME in

an

> individual

> suffering therefrom, comprising administering an effective amount

of a

> corticosteroid.

>

> [0094] Therapeutically effective amounts of minocycline and/or an

> antioxidant

> may also be administered to the subject in conjunction with the

> corticosteroid.

> As will be appreciated, the individual active agents may be

administered

> individually (in two or more separate compositions) or together

(in the

> same

> composition).

>

> [0095] The invention further provides the use of a corticosteroid

in the

> preparation of a medicament for the treatment of CFS/ME. The

medicament

> may be

> formulated for administration in conjunction with minocycline

and/or an

> anti-oxidant. Alternatively, the medicament may comprise

minocycline

> and/or an

> anti-oxidant.

>

> [0096] An example of a suitable corticosteroid is hydrocortisone.

Others

> include dexamethasone and prednisone.

>

> [0097] The invention further provides pharmaceutical compositions

> suitable for

> the treatment of CFS/ME. Thus there is provided a pharmaceutical

> composition

> comprising a therapeutically effective amount of minocycline in

> combination with

> a therapeutically effective amount of a corticosteroid and/or an

> anti-oxidant

> and a pharmaceutically acceptable carrier.

>

> [0098] Also provided is a pharmaceutical composition comprising a

> therapeutically effective amount of a corticosteroid in

combination with

> a

> therapeutically effective amount of minocycline and/or an anti-

oxidant,

> and a

> pharmaceutically acceptable carrier.

>

> [0099] Also provided is a pharmaceutical composition comprising a

> therapeutically effective amount of an anti-oxidant in combination

with a

> therapeutically effective amount of a corticosteroid and/or

minocycline,

> and a

> pharmaceutically acceptable carrier.

>

> [0100] As described above, a particular treatment may be

determined for

> any

> individual based on their expression profile of CFS/ME biomarkers.

For

> example,

> an individual showing upregulation of oxidative stress pathway

genes may

> be

> particularly suitable for treatment with an anti-oxidant. However

it may

> not be

> convenient or necessary to establish a suitable expression profile

before

> beginning treatment. Therefore it may be desirable to administer

one, two

> or

> all three of the treatment types described above to an individual

> affected by

> CFS/ME without recourse to expression profiling.

>

> [0101] Description of the Drawings

>

> [0102] Figure 1 shows a RT-PCR assay of v-ATPase mRNA in PBMCs

from three

> healthy control individuals (lanes 2 to 4) and three patients with

CFS/ME

> (lanes 5 to 7). Lane 1 contains molecular weight markers.

>

> [0103] Figure 2 shows a RT-PCR assay of Defensin alpha 1 mRNA in

PBMCs

> from

> three healthy control individuals (lanes 2 to 4) and three

patients with

> CFS/ME

> (lanes 6 to 8). Lanes 1 and 9 contain molecular weight markers.

>

> [0104] Figures 3 and 4 show Western blots of proteins extracted

from

> PBMCs from

> healthy individuals and individuals suffering from CFS/ME, stained

with

> antibodies against Defensin alpha 1 and Thrombospondin 1

respectively. In

> each

> case, lanes 1 and 12 contain molecular weight markers, lanes 2 to

11

> contain

> samples from patients with CFS/ME, and lanes 13 to 22 contain

samples

> from

> healthy control individuals. Purified protein extract from 2x10<5>

cells

> was

> loaded per lane. Proteins were separated on 10% SDS-PAGE gels and

blotted

> onto

> a nylon membrane.

>

> [0105] Figure 5 shows a SDS-PAGE gel, stained with Coomassie blue,

of the

> same

> samples used to prepare the Western blots shown in Figures 3 and

4. Lanes

> 11 and

> 22 contain molecular weight markers, lanes 1 to 10 contain samples

from

> patients with CFS/ME, and lanes 12 to 21 contain samples from

healthy

> control

> individuals. As in Figures 3 and 4, protein extract from 2x10<5>

cells

> was

> loaded per lane.

>

> [0106] Detailed Description of the Invention

>

> [0107] Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

>

> [0108] CFS is typically diagnosed using the modified CDC criteria

> described by

> Fukuda et al.<2>. All other conditions or diseases which could

explain a

> patient' s symptoms are first excluded. Having done this, CFS/ME is

> diagnosed

> if the patient has been affected by 6 months or longer of

persistent

> relapsing

> or persistent fatigue accompanied by four or more concurrent

symptoms

> including

> impaired memory severe enough to affect normal daily function, sore

> throat,

> tender lymph nodes, muscular or joint pain, new headaches,

unrefreshing

> sleep

> and post-exertional malaise lasting for more than 24 hours. For the

> purposes of

> this specification, individuals satisfying these criteria are

considered

> to be

> affected by CFS/ME.

>

> [0109] Assay methods

>

> [0110] The genes identified in Table 1 provide biomarkers which

may be

> used in

> diagnostic assays to support, confirm, or refute a diagnosis of

CFS/ME.

> With

> the knowledge of this set of genes, it is possible to devise many

methods

> for

> determining a suitable expression profile of one or more CFS/ME

> biomarkers in a

> particular test sample.

>

> [0111] Typically, the method involves contacting expression

products from

> the

> sample with a binding agent capable of binding to an expression

product

> of a

> gene identified in Table 1. The expression product may be a

transcribed

> nucleic

> acid sequence or an expressed polypeptide.

>

> [0112] The transcribed nucleic acid sequence may be mRNA or pre-

mRNA.

> Alternatively, the expression product may also be cDNA produced

from said

> mRNA.

> The binding member may a nucleic acid having a sequence

complementary to

> that of

> the RNA or cDNA which is consequently capable of specifically

binding to

> the

> transcribed nucleic acid or cDNA under suitable hybridisation

conditions,

> e.g.

> by Northern blotting, in situ hybridisation, or Southern blotting.

>

> [0113] Such protocols may use probes of at least about 20-80 bases

in

> length.

> The probes may be of 100, 200, 300, 400 or 500 bases in length or

more.

> Binding assays may be conducted using standard procedures, such as

> described in

> Sambrook et al., Molecular Cloning A Laboratory Manual (New York:

Cold

> Spring

> Harbor Laboratory Press, 1989 or later editions).

>

> [0114] RT-PCR procedures (including quantitative PCR procedures)

may also

> be

> used to analyse the presence or amount of mRNA or precursor mRNA

in a

> given

> sample. A suitable primer having at least 15 to 20 bases

complementary to

> the

> desired mRNA or precursor mRNA sequence will typically be used to

prime

> cDNA

> synthesis. Alternatively a poly-T primer (optionally comprising

one or

> more

> random nucleotides at the 3' end) may be used to prime cDNA

synthesis

> from all

> mRNA in the sample.

>

> [0115] Subsequently, a segment of the cDNA is amplified in a PCR

reaction

> using

> a pair of nucleic acid primers, each typically having at least 15

to 20

> bases

> complementary to the desired RNA sequence. The skilled person will

be

> able to

> design suitable probes or primers based on the publicly available

> sequence data

> for the genes in question (see Table 1 for suitable accession

numbers).

>

> [0116] Where the expression product is the expressed polypeptide,

the

> binding

> member is preferably an antibody raised against or otherwise

specific for

> the

> desired polypeptide, or any other molecule comprising the antigen

binding

> site

> from such an antibody.

>

> [0117] The skilled person will realise that other binding agents

may be

> used as

> appropriate. Suitable agents may include naturally- occurring

ligands and

> receptors for the desired polypeptide, aptamers, etc. For example,

> aptamers

> are nucleic acid molecules (typically DNA or RNA), selected from

> libraries on

> the basis of their ability to bind other molecules. Aptamers have

been

> identified which can bind to other nucleic acids (by means other

than

> conventional -Crick base pairing), proteins, small organic

> compounds,

> and even entire organisms. The binding agent (e.g. a nucleic acid

probe

> or

> antibody) may be fixed to a solid support. The expression products

may

> then be

> passed over the solid support, thereby bringing them into contact

with

> the

> binding agent. Conveniently, the binding agents are immobilised at

> defined,

> spatially separated locations, to make them easy to manipulate

during the

> assay. The solid support may be a glass surface, e.g. a microscope

slide,

> beads, fibre-optics or microarray chip. In the case of beads, each

> binding

> agent may be fixed to an individual bead and they may then be

contacted

> with the

> expression products in solution.

>

> [0118] The sample is generally contacted with the binding agent

(s) under

> appropriate conditions which allow the analyte in the sample to

bind to

> the

> binding agent(s). The fractional occupancy of the binding sites of

the

> binding agent(s) can then be determined.

>

> [0119] Whatever the chosen assay system, there are numerous ways to

> detect

> interaction between the binding agent and the expression product

> (analyte) to be

> determined, either by directly or indirectly labelling " the

analyte or

> binding

> agent, or by using a developing agent to arrive at an indication

of the

> presence

> or amount of the analyte in the sample. A developing agent may be a

> secondary

> binding agent, capable of binding to a complex between analyte and

> primary

> binding agent. For example, if a primary antibody is used as a

binding

> agent,

> the developing agent may be a secondary antibody capable of binding

> either to

> the primary antibody, or to a different epitope on the analyte to

that

> recognised by the primary antibody.

>

> [0120] Typically, the analyte, binding agent or developing agent is

> directly or

> indirectly labelled (e.g. with radioactive, fluorescent or enzyme

labels,

> such as horseradish peroxidase) so that they can be detected using

> techniques

> well known in the art. Directly labelled agents have a label

associated

> with or

> coupled to the agent, Indirectly labelled agents may act on a

further

> species

> to produce a detectable result. Thus, radioactive labels can be

detected

> using

> a scintillation counter or other radiation counting device,

fluorescent

> labels

> using a laser, confocal microscope, etc., and enzyme labels by the

action

> of

> an enzyme label on a substrate, typically to produce a colour

change. In

> further embodiments, the developing agent or analyte is tagged to

allow

> its

> detection, e.g. linked to a nucleotide sequence which can be

amplified in

> a

> PCR reaction to detect the analyte. Other labels are known to those

> skilled in

> the art are discussed below.

>

> [0121] The developing agent (s) can be used in a competitive

method in

> which

> the developing agent competes with the analyte for occupied

binding sites

> of the

> binding agent, or non-competitive method, in which the labelled

> developing agent

> binds analyte bound by the binding agent or to occupied binding

sites.

> Both

> methods provide an indication of the number of the binding sites

occupied

> by the

> analyte, and hence the concentration of the analyte in the sample,

e.g.

> by

> comparison with standards obtained using samples containing known

> concentrations

> of the analyte.

>

> [0122] In alternative embodiments, the analyte can be tagged before

> applying it

> to the support comprising the binding agent.

>

> [0123] There is an increasing tendency in the diagnostic field

towards

> miniaturisation of such assays, e.g. making use of binding agents

(such

> as

> antibodies or nucleic acid sequences) immobilised in small,

discrete

> locations

> (microspots) and/or as arrays on solid supports or on diagnostic

chips.

> These

> approaches can be particularly valuable as they can provide great

> sensitivity

> (particularly through the use of fluorescent labelled reagents),

require

> only

> very small amounts of biological sample from individuals being

tested and

> allow

> a variety of separate assays can be carried out simultaneously.

This

> latter

> advantage can be useful as it provides an assay employing a

plurality of

> analytes to be carried out using a single sample. Examples of

techniques

> enabling this miniaturised technology are provided in WO84/01031,

> WO88/1058,

> WO89/01157, WO93/8472, WO95/18376/ WO95/18377, WO95/24649 and EP 0

373

> 203 A.

>

> [0124] Other methods which do not rely on labelling techniques may

also

> be used

> to detect interaction between binding agent and reporter molecule,

> including

> physical methods such as surface plasmon resonance, agglutination,

light

> scattering or other means.

>

> [0125] Expressed nucleic acid (mRNA, pre-mRNA) can be isolated

from the

> cells

> using standard molecular biological techniques. The expressed

nucleic

> acid

> sequences corresponding to the gene or genes of Table 1 can then be

> amplified

> using nucleic acid primers specific for the expressed sequences in

a PCR,

> e.g.

> real time PCR, multiplex PCR, etc. The skilled person will be able

to

> select

> or design a suitable reaction type and protocol depending on, e.g.

the

> number

> and particular combination of genes to be analysed. If the isolated

> expressed

> nucleic acid is mRNA, this can be converted into cDNA for the PCR

> reaction using

> standard methods.

>

> [0126] The primers may conveniently introduce a label into the

amplified

> nucleic

> acid so that it may be identified. Ideally, the label is able to

indicate

> the

> relative quantity or proportion of nucleic acid sequences present

after

> the

> amplification event, reflecting the relative quantity or proportion

> present in

> the original test sample. For example, if the label is fluorescent

or

> radioactive, the intensity of the signal will indicate the

relative

> quantity/proportion or even the absolute quantity, of the expressed

> sequences.

> The relative quantities or proportions of the expression products

of each

> of the

> genes of Table 1 may be used to establish a particular expression

profile

> for

> the test sample.

>

> [0127] Other methods for detection of nucleic acid expression

products

> may also

> be used, such as in situ hybridisation, Northern blot, etc.

Likewise,

> protein

> expression products may be detected by any suitable technique.

> Immunological

> techniques are particularly preferred, in which antibodies

specific for

> the

> particular polypeptide gene product (s), are used as binding

agents,

> although

> other binding agents such as receptors or ligands capable of

binding to

> the

> proteins of interest may be employed.

>

> [0128] In some embodiments, protein expression products from the

sample

> under

> test are immobilised on a solid phase and contacted with a binding

agent

> specific for one or more of the proteins of Table 1 under

appropriate

> conditions

> which allow binding between the protein and the binding agent. The

amount

> of

> the binding agent found at the surface is then determined. For

example,

> the

> binding agent may be directly labelled. Alternatively, the

immobilised

> antibody

> may be contacted with a labelled developing agent capable of

binding to

> the

> primary antibody. Examples of this type of assay include Western

> blotting, and

> certain ELISA (enzyme-linked immunosorbent assay) techniques.

>

> [0129] In other embodiments, a binding agent is immobilised on a

solid

> phase

> and contacted with the sample under suitable conditions to allow

binding

> to take

> place. The fractional occupancy of the binding sites of the

binding agent

> (s)

> can then be determined either by directly or indirectly labelling

the

> analyte or

> by using a developing agent or agents to arrive at an indication

of the

> presence

> or amount of the analyte in the sample.

>

> [0130] An example of this type of assay is an antibody sandwich

assay

> (e.g. an

> ELISA), which employs two antibodies each capable of binding to a

> different

> site on the biomarker protein. The first is immobilised on a solid

phase

> for use

> as the binding agent. After contact with the analyte, the second

antibody

> is

> used to detect complexes between the first antibody and analyte.

> Whichever

> method is chosen, it is important that the assay provides a read-

out of

> the

> level of expression of the biomarker genes which allows results

from

> different

> individuals to be compared reliably with one another. By way of

example,

> the

> level of a particular expression product may be determined as a

> proportion of

> the total expression products found in the sample. Alternatively,

the

> level of

> a particular expression product may be determined in relation to

the

> level of

> expression of a control gene such as a housekeeping gene, or the

like.

> Alternatively, it may be convenient to determine the absolute

amount of a

> particular expression product, e.g. by comparison with known

standards.

> The

> skilled person will be capable of designing a suitable protocol

for any

> given

> assay method, and will also be aware of other suitable embodiments.

>

> [0131] Antibodies

>

> [0132] It has been shown that fragments of a whole antibody can

perform

> the

> function of binding antigens. The term " antibody " is therefore used

> herein to

> encompass any molecule comprising the binding fragment of an

antibody,

> and the

> term binding agent and binding site should be construed

accordingly.

> Examples

> of binding fragments are (i) the Fab fragment consisting of VL,

VH, CL

> and CHl

> domains; (ii) the Fd fragment consisting of the VH and CHl

domains; (iii)

> the

> Fv fragment consisting of the VL and VH domains of a single

antibody;

> (iv) the

> dAb fragment (Ward, E.S. et al., Nature 341, 544-546 (1989)) which

> consists of a VH domain; (v) isolated CDR regions; (vi) F (ab ') 2

> fragments,

> a bivalent fragment comprising two linked Fab fragments (vii)

single

> chain Fv

> molecules (scFv), wherein a VH domain and a VL domain are linked

by a

> peptide

> linker which allows the two domains to associate to form an antigen

> binding site

> (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA,

85,

> 5879-5883,

> 1988). In preferred embodiments the binding agent comprises a

single

> antigen

> binding site specific for the analyte, i.e. a monovalent antibody

or

> antibody

> fragment.

>

> [0133] Pha rmaceutical compositions Pharmaceutical compositions as

> described in

> this specification typically comprise, in addition to one or more

> suitable

> active agents, a pharmaceutically acceptable excipient, carrier,

buffer,

> stabiliser or other materials well known to those skilled in the

art.

> Such

> materials should be non-toxic and should not interfere with the

efficacy

> of the

> active ingredient. The precise nature of the carrier or other

material

> may

> depend on the route of administration, e.g. oral, intravenous,

cutaneous

> or

> subcutaneous, nasal, intramuscular, intraperitoneal routes.

>

> [0134] Pharmaceutical compositions for oral administration may be

in

> tablet,

> capsule, powder or liquid form. A tablet may include a solid

carrier such

> as

> gelatin or an adjuvant. Liquid pharmaceutical compositions

generally

> include a

> liquid carrier such as water, petroleum, animal or vegetable oils,

> mineral oil

> or synthetic oil.

>

> [0135] Physiological saline solution, dextrose or other saccharide

> solution or

> glycols such as ethylene glycol, propylene glycol or polyethylene

glycol

> may be

> included.

>

> [0136] For intravenous, cutaneous or subcutaneous injection, or inj

> ection at

> the site of affliction, the active ingredient will be in the form

of a

> parenterally acceptable aqueous solution which is pyrogen-free and

has

> suitable

> pH, isotonicity and stability. Those of relevant skill in the art

are

> well able

> to prepare suitable solutions using, for example, isotonic

vehicles such

> as

> Sodium Chloride Inj ection, Ringer ' s Inj ection, Lactated

Ringer ' s

> Inj

> ection. Preservatives, stabilisers, buffers, antioxidants and/or

other

> additives may be included, as required.

>

> [0137] Whether it is a polypeptide, antibody, peptide, nucleic acid

> molecule,

> small molecule or other pharmaceutically useful compound that is

to be

> given to

> an individual, administration is preferably in a " prophylactically

> effective

> amount " or a " therapeutically effective amount " (as the case may

be,

> although

> prophylaxis may be considered therapy), this being sufficient to

show

> benefit

> to the individual. The actual amount administered, and rate and

> tir[alpha]e-

> course of administration, will depend on the nature and severity

of what

> is

> being treated. Prescription of treatment, e.g. decisions on dosage

etc,

> is

> within the responsibility of general practitioners and other

medical

> doctors,

> and typically takes account of the disorder to be treated, the

condition

> of the

> individual patient, the site of delivery, the method of

administration

> and other

> factors known to practitioners. Examples of the techniques and

protocols

> mentioned above can be found in Remington' s Pharmaceutical

Sciences,

> 20th

> Edition, 2000, pub. Lippincott, & Wilkins.

>

> [0138] Alternatively, targeting therapies may be used to deliver

the

> active

> agent more specifically to certain types of cell, by the use of

targeting

> systems such as antibody or cell specific ligands. Targeting may be

> desirable

> for a variety of reasons; for example if the agent is unacceptably

toxic,

> or if

> it would otherwise require too high a dosage, or if it would not

> otherwise be

> able to enter the target cells.

>

> [0139] A composition may be administered alone or in combination

with

> other

> treatments, either simultaneously or sequentially dependent upon

the

> condition

> to be treated.

>

> [0140] Examples A group at the University of UIm, Germany, has

recently

> suggested that a pentapeptide (QYNAD) with Na<+> channel-blocking

> function could

> be a biological marker of certain inflammatory and immunological

> disorders of

> the nervous system<16>.

>

> [0141] The inventors asked whether or not the pentapeptide

identified by

> the

> German group<16> might play a role in CFS. Samples of serum were

sent to

> the

> University of UIm for analysis. The 15 samples included 5 normal

> controls, 5

> patients with CFS and 5 disease controls including two patients

with MS.

> Samples were numbered 1-15 and the German group were not informed

what

> the

> samples were, or which samples were which, until the experiment was

> concluded.

>

> [0142] When the code was broken, the results showed that the

disease

> control

> group had levels of the pentapeptide which were 2.3X those of the

normal

> controls (similar to the published data) and the CFS samples had

levels

> which

> were 3X higher than the healthy controls.

>

> [0143] Thus, there are measurably higher amounts of the

pentapeptide in

> patients with CFS compared with healthy controls. Although the

> pentapeptide may

> not be specific to CFS (as high levels are also found in other

> disorders), an

> assay for the peptide could be used as part of the differential

diagnosis

> of CFS.

>

> [0144] The German group was unable to identify an endogenous gene

which

> encodes

> the pentapeptide. The inventors carried out NCBI BLAST and

> EMBL-Heidelberg

> Bioccelerator amino acid alignments for the pentapeptide QYNAD. A

total

> of one

> hundred alignment hits were found. Of these, only nine showed 100%

> similarity

> over the five amino acids - five of those were human. The amino

acid

> searches

> were followed by NCBI BLAST searches using the GenBank Accession

and gi

> numbers

> for each of the five human amino acid to determine their origins,

> references

> and nucleotide sequences. A number of cloned nucleotide sequences

were

> found

> and when these were run through the nucleotide databases, only one

clone

> showed

> full-length homology to any human gene. This gene was a human ion-

channel

> gene

> - the vacuolar proton pump H<+>-ATPase (v-ATPase). Remarkably,

when the

> human

> gene amino acid sequence was compared with the original QYNAD

> pentapeptide it

> was discovered that the relevant part of the human ion channel

encodes

> the

> sequence QYMAD.

>

> [0145] The inventors next asked whether the v-ATPase represents a

> candidate gene

> for a diagnostic test for CFS. RT-PCR using primers specific for

the

> v-ATPase

> was performed on cDNA prepared from mRNA from PBMCs from CFS

patients and

> healthy controls. As shown in Figure 1, the patient samples have a

> significantly higher level of v-ATPase mRNA than the healthy

controls.

> Thus the

> v-ATPase gene appears to represent a genuine biomarker for CFS/ME.

>

> [0146] The vATPase is known to be involved in regulation of a

number of

> metabolic functions which are deranged in CFS/ME. vATPase

upregulation

> could

> therefore provide an explanation for a number of the symptoms

observed.

> For

> example, increased vATPase activity could explain the intracellular

> acidosis in

> exercising muscles, chest pain (syndrome X), altered

neurotransmitter

> (dopamine) function and abnormal regulation of hypothalamic

hormones. In

> addition, it could explain the increased energy expenditure and

fatigue

> associated with the condition. Taken together, this suggests that

the

> vATPase is

> not only a marker for the condition, but is a realistic target for

> intervention

> therapy.

>

> [0147] Other biomarkers for CFS/ME

>

> [0148] The inventors went on to examine whether the increase in

vATPase

> expression was confirmed by microarray analysis. Such analysis

provides

> the

> opportunity to examine the differential expression of mRNA from a

very

> large

> number of genes. Surprisingly, the results of the analysis not only

> confirmed

> their earlier findings regarding the vATPase gene, but also

identified

> differences in the level of expression of key genes in the PBMC of

> patients with

> CFS/ME and control subj ects, giving an insight into the

biochemical

> pathways

> which are involved in this disorder.

>

> [0149] Microarray results have been verified by western blot

analysis and

> RT-PCR

> assay. A number of genes, in addition to v-ATPase, were

significantly

> up/downregulated and identified as suitable biomarkers for the

disorder.

>

> [0150] Advances in genome sequencing and automated chip

manufacture have

> made

> DNA chip or microarray technology readily available<25>. This

technology

> allows

> simultaneous differential expression profiling from a very large

number

> of genes

> in tissue samples of CFS/ME patients and controls. A recent report

from

> Vernon

> et al (2002), described a CFS biomarker search in PBMC using a DNA

chip

> array

> assay which included 1, 764 genes. In the study reported here, RNA

> isolated

> from PBMC, was assayed using Affymetrix genome-wide chips (HG-U133

> arrays)

> which included 30,000 gene sequences.

>

> [0151] Using DNA microarray analysis of whole human genome, gene

> transcriptional signatures were compared in the PBMC of eight male

> patients with

> CFS and seven age-matched male healthy controls. An additional

cohort of

> fourteen patients with CFS and age and sex matched controls was

recruited

> for

> RT-PCR and western blot assays in order to verify the microarray

data.

> Analysis

> of the microarray data was performed as described previously

(Breitling

> R.

> Armengaud P. Amtmann A. Herzyk P. Rank products: A simple, yet

powerful,

> new

> method to detect differentially regulated genes in replicated

microarray

> experiments. FEBS Letters 2004; 573 (1-3): 83-92; Breitling R.

Amtmann A.

> Herzyk P. Iterative Group Analysis (iGA): A simple tool to enhance

> sensitivity

> and facilitate interpretation of microarray experiments. BMC

> Bioinformatics

> 2004; 5: (pp 8p)).

>

> [0152] Genes which are significantly upregulated in CFS/ME patients

> compared to

> healthy controls are detailed in Table 6, ranked according to

their RP

> values.

> It is considered that any of these genes may be used as biomarkers

for

> CFS/ME.

> Particularly preferred marker genes are detailed in Table 1.

>

> [0153] Further genes, including prostaglandin D2 synthase and T-

cell

> receptors

> alpha, beta, gamma and delta are found to be downregulated in CFS

> patients

> compared to normal controls. Prostaglandin D2 synthase (NCBI

accession

> no. BC

> 005939, UniGene Hs. 446429) is considered to be a good candidate

for a

> CFS/ME

> biomarker, because it is known to be involved in sleep regulation;

> patients with

> CFS frequently suffer from sleep reversal and fatigue associated

with

> lack of

> sleep. However, data for other downregulated genes is not shown

here. In

> general, genes which are upregulated in the disease state are

considered

> to be

> better biomarkers for diagnostic tests etc. than genes which are

> downregulated

> because the potential for false-positive tests is significantly

higher

> when

> using genes which are underexpressed in the disease state.

>

> [0154] Iterative group analysis of the differentially expressed

genes

> indicate

> that in CFS, there is a shift of immune response with preferential

> antigen

> presentation to MHC class Il receptors and downregulation of T-

cell

> receptor-[alpha], increased cell membrane prostaglandin-

endoperoxide

> synthase

> activity with downstream changes in oxygen transport and also

activation

> of the

> guanyl cyclase and caspase pathways of cellular apoptosis. Another

set of

> genes

> was identified which are involved in the immediate response to

infection,

> particularly by intracellular parasites. The particular genes

involved in

> each

> of these pathways are identified in Tables 2 to 5. In each of

these key

> pathways,

> the hub genes were higher ranked in the analysis compared to the

network

> genes.

>

> [0155] Functional changes produced by altered gene regulation may

explain

> the

> mechanism of fatigue and offer a rational basis for targeted

> pharmacotherapy in

> CFS.

>

> [0156] Table 1 - Biomarker Genes for CFS/ME

>

> [0157]

> <EMI ID=36.1>

>

>

> [0158]

> <EMI ID=37.1>

>

> [0159] Table 2: Genes involved in defence against infection by

> intracellular

> pathogens

>

> [0160]

> <EMI ID=37.2>

>

> [0161]

> <EMI ID=38.1>

>

> [0162] Table 3: Genes involved in oxidative stress response

>

> [0163]

> <EMI ID=38.2>

>

> [0164] Table 4: Genes involved in apoptosis

>

> [0165]

> <EMI ID=38.3>

>

> [0166] Table 5: Genes involved in antigen presentation and immunity

>

> [0167]

> <EMI ID=38.4>

>

> [0168]

> <EMI ID=39.1>

>

> [0169] NCBI accession numbers refer to the UniGene database, build

no.

> 177,

> released 22 December 2004.

>

> [0170] It is clear from this data that significant differences in

the

> expression

> of a number of genes can be seen in PBMC samples from patients

with CFS

> and

> healthy controls. We have verified that the DNA microarray assay

is valid

> by

> confirming the results by RT-PCR and western blot analyses. This

is the

> first

> time that a reproducible biochemical lesion has been seen in

patients

> with CFS.

> We propose that bioassays of the significantly over-expressed

genes could

> be

> used as diagnostic biomarkers for CFS to aid in the differential

> diagnosis of

> the condition.

>

> [0171] In order to confirm the relevance of the genes identified

in the

> microarray experiments, Western blot and RT-PCR assays have been

> performed to

> analyse the expression of selected genes in samples from patients

and

> controls

> different to those studied in the microarray analysis. The results

verify

> that

> these genes may be used as potential biomarkers to support the

clinical

> diagnosis of CFS and identify suitable candidates for treatment

trials.

>

> [0172] Figures 2 to 5 show upregulation of mRNA and/or proteins

from

> various of

> these genes in PBMCs from patients suffering from CFS/ME, as

compared to

> age-

> and sex-matched healthy controls. Figure 2 shows RT-PCR analysis of

> Defensin

> alpha 1, Figures 3 and 4 show Western blot analysis of Defensin

alpha 1

> and

> Thrombospondin 1 respectively. In all cases, there is significant

> upregulation

> of the protein or mRNA in the disease group compared to the control

> group.

> Figure 6 shows a duplicate of the SDS-PAGE gel used for the

Western blot,

> stained with Coomassie blue. This confirms that the observed

differences

> in

> protein levels between the disease and control groups are not due

to

> unequal

> loading of total protein on the gels.

>

> [0173] Discussion

>

> [0174] Previous reports have hypothesised that CFS is a form of

> channelopathy -

> a disorder of membrane ion channels<9>' <11>' <13>. There are

several

> reports in

> the literature which we believe strengthen the hypothesis that the

> vacuolar H+

> ATPase plays a pathogenic role in CFS.

>

> [0175] Local anaesthetics, which are known to act on ion channels,

have

> an

> adverse effect on patients with CFS/ME. It has been demonstrated

also,

> that in

> some patients with CFS/ME, there are morphological changes to the

red

> blood

> cells<19>. Remarkably, a study by Nishiguchi et al<20>, has

demonstrated

> that

> the local anaesthetic lidocaine can induce reversible

morphological

> transformation of human red blood cells and that this change is

mediated

> by the

> activation of vacuolar H+ ATPase. In addition, Li et al<21a> have

shown

> that

> the gene is involved in iron binding in red blood cells.

>

> [0176] The ion channel gene is a member of the vacuolar H+ ATPase

proton

> transporting gene family<21>'<22>'<23>. This family of genes is

directly

> involved with the phosphocreatine-dependent glutamate uptake by

synaptic

> vesicles<24>. The gene is responsible for vesicle

docking/exocytosis

> during

> neurotransmiter release<25> and is a major constituent of synaptic

> vesicles

> associated with intracellular membrane structures<26>. We have

> demonstrated,

> using <1>H MRS that there is a perturbation of the choline/creatine

> balance in

> the CNS (Condon et al<17>, Chaudhuri et al<42>). This finding has

been

> corroborated by Puri et al<18>. As stated above, this type of gene

is

> directly

> involved in the creatine pathways. We have previously demonstrated

that

> patients with CFS have low body- potassium levels<9>. et

al<27>

> have

> shown a relationship between potassium depletion and up-regulation

of

> H+-ATPase.

>

> [0177] As stated above, viruses have often been associated with

CFS.

> Virus

> entry into cells may be mediated by H+ATPase<28>'<29>'<30>. In

addition

> to viral

> infection affecting neurotransmitter function<7>, there is a large

body

> of

> evidence to show that the vacuolar H+-ATPase is also

> invoked<31>'<32>'<33,34>'

> <35>'<36>'<37>'<38, 39.>

>

> [0178] It is clear from the above data that significant

differences in

> the

> expression of a number of genes can be seen in PBMC samples from

patients

> with

> CFS and healthy controls. This is the first time that a

reproducible

> biochemical lesion has been seen in patients with CFS We propose

that

> bioassays

> of the significantly over-expressed genes (below) could be used as

> diagnostic

> biomarkers for CFS to aid in the differential diagnosis of the

condition.

>

> [0179] Materials and Methods

>

> [0180] Study Subjects

>

> [0181] Patients with CFS were diagnosed with reference to the 1994

Fukuda

> definition. All seven patients were male, aged between 18 and 54

years

> (mean

> 36), and were not on medication. Healthy control subj ects were

male,

> aged

> between 22 and 58 years (mean 34). In addition to the 8 patients

and

> seven

> control subjects used for the DNA chip assays, an additional

fourteen

> patients

> and controls were used to confirm the chip assay results by RT-PCR

and

> western

> blot assays. Informed consent and ethical approval were obtained.

>

> [0182] Isolation of peripheral blood mononuclear cells (PBMC) for

protein

> purification

>

> [0183] Venous blood samples were drawn from patients who fulfilled

the

>

> [0184] Holmes and Fukuda criteria for ME/CFS, and from healthy

> individuals. The

> procedure for isolating PBMC was started immediately and finished

within

> 2h of

> sampling. EDTA treated whole blood was diluted 1: 1 with phosphate

> buffered

> saline. Two volumes of blood were overlaid on one volume of

Histopaque

> -1077

> (Sigma Diagnostics) and centrifuged at 20[deg.]C AT 50Og for 30

min. The

> PBMC

> interface was removed and washed twice with phosphate buffered

saline and

> centrifuged. The pellets were resuspended in phosphate buffered

saline,

> an

> aliquot removed and counted in red blood cell lysis buffer (155Mm

NH4CL,

> 10Mm

> NaHCO3, pH7.4, 0.1mM EDTA). The PBMC were centrifuged once more

> (20[deg.]C,

> 500g, 10min). The PBMC were then aliquoted into micortubes

equivalent to

> 5x105

> cells per tube, centrifuged and stored as dry pellets at -80[deg.]

C.

>

> [0185] Western Blot Analysis

>

> [0186] PBMC pellets were resuspended in sample reducing buffer (ImI

> Glycerol,

> 0.5ml [beta]-mercaptoethanol, 3ml 10% SDS, 1.25ml IM Tris-HCL Ph

6.7),

> boiled

> for 5 min. The lysates were loaded onto a 10% PAGE gel, each track

> equivalent

> to 2x 10 5 PBMC. The PAGE gel was assessed for equal protein load

by

> coomassie

> stain. The gel then electrophoretically transferred onto

nitrocellulose

> PVDF

> membrane (Biorad) for 2 hours. The blots were blocked for non

specific

> binding

> with 10% normal goat serum for 30 min, probed with a mouse Mab to

human

> defensinl-3, (Hycult, Netherlands), mouse Mab to human

thrombospondin

> (Sigma-Aldrich Inc) and a mouse Mab to Chondroitin Sulphate

Proteoglycan

> (USBiological MA USA), at dilutions of 1/100, 1/1000, 1/1000

respectively

> in

> TBS 0.05% Tween 20 for 2 hours at RT. The protein was detected

after

> subsequent

> incubation with alkaline phosphatase conjugate Goat anti Mouse IgG

> (1/1000

> final dilution) ( Immunoresearch Laboratories PA USA). The

> reactions

> were detected using SIGMA FAST BCIP/NBT).

>

> [0187] Development of the PCR assay for the CFS gene.

>

> [0188] Oligonucleotide primers which span a specific epitope

within the

> CFS gene

> were chosen and tested by RT-PCR. RNA from blood samples from

patients

> with CFS

> and appropriate controls were RT-PCR amplified and PCR amplicons

> quantitated by

> gel documentation system software.

>

> [0189] RNA Isola tion for RT-PCR

>

> [0190] Total RNA was isolated from peripheral blood mononuclear

cells

> (PBMC)

> using the Promega RNAgents Total RNA Isolation System.

>

> [0191] Venous blood was collected in standard EDTA blood tubes and

RNA

> purified

> using the method of Chomczynski and Sacchi. White blood cell

pellets were

> homogenised by hand in an appropriate volume of denaturing solution

> (guanidinium

> thiocyanate, 4M; sodium citrate, 25mM; N-laurolyl sarcosine, 0.5%;

> 2-mercaptoethanol, 0.1M, in distilled water adjusted to pH 7.0). To

> homogenate

> (ImI), sodium acetate (100[mu]l, 2M, pH 4.0), citrate-buffered

(0.1M, pH

> 4.3) phenol

>

> [0192] { lml) and chloroform: isoamyl alcohol (49: 1, 200[mu]l)

were

> sequentially added. The resulting mixture was treated in a vortex

mixer

> (Fisons

> Scientific Equipment, Whirlimixer) for 10 seconds then

incubated on

> ice for

> 15 minutes. Samples were then centrifuged (12, 000g, 20 minutes,

> 4[deg.]C) and

> the upper aqueous layer pippetted into to a fresh tube. After

addition of

> ice

> cold isopropanol (lml), RNA precipitated from the mixture during a

thirty

> minute period on dry ice. The mixtures were then centrifuged (12,

000g,

> 20

> minutes, 4[deg.] C) and the supernatant discarded. The pellet was

> dissolved in

> denaturing solution (300[mu]l) and transferred to a

microcentrifuge tube (

> 1.5ml, Axygen). Ethanol (absolute, 600[mu]l) was added to each

> microcentrifuge

> tube, samples were incubated on dry ice for 30 minutes, and then

> centrifuged (

> 1160Og, 20 minutes, 4[deg.]C). The resulting pellet were then

washed

> twice in

> ethanol (70% aqueous solution) then lyophylised (using a Hetosicc

> freeze-drier

> and a JAVAC high vacuum pump DD-75). The freeze-dried RNA was

> re-suspended in

> sterile distilled water (60[mu]l) and stored at -70[deg.]C until

required.

>

> [0193] Quantifica tion and examination of RNA

>

> [0194] RNA solution (prepared as described above, 2[mu]l) was

added to

> distilled

> water (98[mu]l) to produce a 1: 50 dilution. The optical density

of the

> sample was read at 260nn[alpha] and 280nm using a 50[mu]l

ultraviolet

> cuvet.

> The absorbance ratio 260/280nm measured at these wavelengths

indicates

> the

> purity of the RNA. The absolute concentration of RNA was estimated

using

> the

> following equation:

>

> [0195] Optical Density260nm x 40 x dilution = RNA concentration

([mu]g/ml)

>

> [0196] To examine the quality, 5[mu]l of RNA was added to 5[mu]l

of the

> gel

> marker Orange G (1% Orange G dye in 50% glycerol, 50% 2x TBE).

This was

> heated

> to 70[deg.]C for 3 minutes, then electrophoresed through a

horizontal 1%

> agarose gel in 1 x TBE (10x TBE = 0.089M tris (hydroxymethyl) -

> methylamine,

> 0.089M boric acid, 0.025M disodium EDTA, pH 8.3). The gel was then

> stained for

> 30 minutes in ethidium bromide (0.5 mg/ml) in TBE, de-stained in

water

> and

> visualised under medium wave (320nm) ultraviolet light.

>

> [0197] cDNA synthesis

>

> [0198] Two micrograms of RNA were added to 1[mu]l oligo (dT) 12-18

(

> 500[mu]g/ml, Roche) and the volume made up to 11[mu]l with sterile

> distilled

> water.

>

> [0199] This was incubated (70[deg.]C, 10 minutes) to allow the

oligo (dT)

> to

> bind to the poly-A tail of the RNA, and then chilled on ice. To the

> reaction

> mixture, 4[mu]l of 5x First Strand Buffer, 2[mu]l of 0. IM DTT, 1

[mu] l

> of 10mM

> dNTP mix (10mM each dATP, dGTP, dCTP, dTTP; Amersham Pharmacia

Biotech),

> 1[mu]l of sterile distilled water and 1[mu]l of

>

> [0200] Superscript II (Gibco-BRL® Life Technologies) were added.

This

> was

> incubated first at 50[deg.]C for Ihr, then 70[deg.]C for 15

minutes to

> inactivate the reaction. A negative cDNA control (2[mu]l of

distilled

> water)

> was included in each cDNA synthesis to confirm that the reaction

mix was

> not

> contaminated.

>

> [0201] Polymerase Chain Reaction To 2[mu]l of cDNA, 10[mu]l of 10x

> magnesium-free buffer, 10[mu]l of 2.5mM dNTPs (2.5mM each dATP,

dGTP,

> dCTP,

> dTTP), 6[mu]l MgCl2 (25mM) and 1[mu]l each of the appropriate 5'

and 3 '

> primers (0.5[mu]g/[mu]l) were added. The volume was made up to 99.8

[mu]l

> using

> sterile distilled water and 0.2[mu]l of Taq DNA polymerase

(Promega) was

> added.

> The PCR reaction (35 cycles) was carried out on a Techne Genius

> thermocycler.

>

> [0202] Primers used to amplify vATPase were 5 ' -etc gtg ace tgt

tac tgc

> tg - 3

> ' and 5 ' -aag taa cca agt cca etc ca-3 '. Primers for Defensin 1

were 5

> ' -caa

> gag ctg atg agg ttg ct-3 ' and 5 ' -gaa ggt aca gga gta ata gc-3 '.

>

> [0203] Thirty microlitres of PCR product was added to 5[mu]l of

orange G

> and

> electrophoresed through a horizontal 2-3% agarose gel in 1 x TBE

at 100

> volts,

> until the dye front had migrated a minimum of 10cm. On each gel, 3

[mu]l

> of a 123

> base pair DNA ladder in 5[mu]l of Orange G was included as a size

marker

> for

> comparison with PCR product bands. The gel was then stained for 30

> minutes in

> ethidium bromide (0.5 mg/ml) in TBE, destained in water and

visualised

> under

> medium wave (320nm) ultraviolet light.

>

> [0204] Gene expression was analysed by measuring the band density

for

> each

> amplicon. Band densities were measured using the Herolab EASY Plus

> computer

> automated image analysis system. True comparisons in gene

expression

> between

> samples were enabled by comparing results of densitometry for the

> experimental

> genes against the housekeeping gene (abl - tyrosine kinase) band

> densities.

> References

>

> [0205] 1. Chaudhuri A, Behan PO. Neurological dysfunction in

chronic

> fatigue

> syndrome. J Chr Fatigue Synd 2000; 6 (3/4): 51-68

>

> [0206] 2. Fukuda K, Strauss SE, Hickie I, et al. The chronic

fatigue

> syndrome:

> a comprehensive approach to its definition and study. Ann Intern

Med

> 1994;

> 121: 953-59

>

> [0207] 3. The National Task Force Report on CFS/ME (30 September

1998).

>

> [0208] 4. Chaudhuri A, Behan WMH, Behan PO. Chronic fatigue

syndrome.

> Proc R

> Coll Physicians Edinb 1998: 28: 150-63 5. Chaudhuri A, Gow J,

Behan PO.

> Systemic viral infections and chronic fatigue syndrome: current

concept

> and

> recent advances. In: Abramsky O, D (eds). Brain disease:

> Therapeutic

> strategies and repair. London: Dunitz 2001 (in press)

>

> [0209] 6. de Ia Torre JC, Borrow P, Oldstone MBA. Viral

persistence and

> disease:

> cytopathology in the absence of cytolysis. Br Med Bull

>

> [0210] 1991; 47: 838-51

>

> [0211] 7. Gies U, Bilzer T, Stitz L, Staiger JF. Disturbance of the

> cortical

> cholinergic innervation in Borna disease prior to encephalitis.

Brain Pa

> thology 1998; 8: 39-48 8. Kress M, Fickenscher H. Infection by

human

> varicella-zoster virus confers norepinephrine sensitivity to

sensory

> neurons

> from rat dorsal root ganglia. FASEB Journal 2001; 15: 1037-43

>

> [0212] 9. Chaudhuri A, WS, Behan PO. Arguments for a role of

> abnormal

> ionophore function in chronic fatigue syndrome. In: Yehuda S,

Moltofsky

> DI

> (eds). Chronic fatigue syndrome. New York, Plenum Press 1997; 119-

30

>

> [0213] 10. Goncealves PP, Meireles SM, Neves P, Vale MGP. Ca<2+>

> sensitivity of

> synaptic vesicle dopamine, gamma-aminobutyric acid and glutamate

> transport

> system. Neurochemical research 2001; 26: 75-81

>

> [0214] 11. Gow JW, McGarry F, Behan WMH, Simpson K, Behan PO.

Molecular

> analysis

> of cell membrane ion channel function in chronic fatigue syndrome

> (Abstract).

> International Meeting on Chronic Fatigue Syndrome, Dublin 1994. 12.

> Ackerman MJ,

> Clapham DE. Ion channels-basic science and clinical diseases. N

Engl J

> Med

> 1997; 336: 1575-86

>

> [0215] 13. Chaudhuri A, WS, Pearn J, Behan PO. Symptoms of

chronic

> fatigue syndrome are due to abnormal ion channel function. Medical

> Hypotheses

> 2000; 54: 59-63.

>

> [0216] 14. Pearn J. Chronic ciguatera: one organic cause of the

chronic

> fatigue

> syndrome. J Chr Fa tigue Synd 1996; 2: 29-34

>

> [0217] 15. Bidard JN, Viverberg HPM, Frelin C. Ciguatoxin is a

novel type

> of

> Na<+> channel toxin. J Biol Chem 1984; 259: 8353-57 16. Brinkmeier

H,

> Aulkemeyer P, Wollinsky KH, Rudel R. An endogenous pentapeptide

acting as

> a

> sodium channel blocker in inflammatory autoimmune disorders of the

> central

> nervous system. Nature Med 2000; 6: 808-11.

>

> [0218] 17. Condon BR, Chaudhuri A, Hadley DN, Brennan D, (2002),

> " Increased

> levels of choline containing compounds found in the basal ganglia

of

> patients

> with chronic fatigue syndrome " in Proc Intl. Soc. Mag. Reson. Med.

10.

>

> [0219] 18. Puri BK, et al, (2002), " Relative increase in choline

in the

> occipital cortex in chronic fatigue syndrome " Acta Psychiatr Scand

106

> (3):

> 224-226.

>

> [0220] 19. Simpson LO. (1992) " The role of nondiscocytic

erythrocytes in

> the

> pathogenesis of myalgic encephalomyelitis/chronic fatigue

syndrome " In

> Hyde BM,

> Goldstein, Levine P (eds). The clinical and scientific basis of

myalgic

> encephalomyelitis/chronic fatigue syndrome. The Nightingale

Research

> Foundation, Canada. 597-605.

>

> [0221] 20. Nishiguchi E, Hamada N, Shindo J. (1995), " Lidocaine

action

> and

> conformational changes in cytoskeletal protein network in human

red blood

> cells "

> Eur J Pharmacol, 286 (1): 1-8. 21. Forgac, M. Structure and

properties of

> the vacuolar (H<+>) - ATPases-. J. Biol. Chem. 274 (19), 12951-

12954

> (1999).

> 21a. Li, C-Y., Watkins, J.A., Hamazaki, S., Altazan, J. D. &

Glass, J.

> Iron binding, a new function for the reticulocyte endosome H+-

ATPase.

> Biochemistry. 34 (15), 5130-5136 (1995). 22. s, T. H. &

Forgac,

> M. Structure, function and regulation of the vacuolar (H<+>) -

ATPase.

> Annu.

> Rev. Cell Dev. Biol 13, 779-808

>

> [0222] (1997).

>

> [0223] 23. , N. & Harvey, W. R. Vacuolar and plasma membrane

> proton-

> adenosinetriphosphatases. Physiol. Rev. 79 (2), 361-385 (1999).

>

> [0224] 24. Xu CJ, Klunk WE, Kanfer JN, et al, (1996),

Phosphocreatine-

> dependent glutar[alpha]ate uptake by synaptic vesicles " J

Biological

> Chemistry,

> 271 (23): 13435-13440.

>

> [0225] 25. Siebert A, Lottspeich F, N, Betz H (1994).

Purification

> of

> the synaptic vesicle-binding protein physophilin.

>

> [0226] Identification as 39-kDa subunit of the vacuolar H+-

ATPase. J.

> Biological Chemistry. 269 (45): 28329-28334.

>

> [0227] 26. Brochier G, Morel N (1993). The same 15kDA proteolipid

subunit

> is a

> constituent of two different proteins in Torpedo, the acetycholine

> releasing

> protein and the vacuolar H+ ATPase. Neurochem. International. 23

(6):

> 525-539.

>

> [0228] 27. , M., Capasso, G., Agulian, S., Giebisch, G. &

Unwin, R.

> The relationship between distal tubular proton secretion and

dietary

> potassium

> depletion: evidence for up-regulation of H+- ATPase. Nephrol.

Dial.

> Transplant. 14 (6), 1435-1440 (1999).

>

> [0229] 28. Hansen, J., Qing, K. & Srivastava, A. Adeno-associated

virus

> type 2-medicated gene transfer: Altered endocytic processing

enhances

> transduction efficiency in murine fibroblasts. J. Virol. 75 (9),

4080-4090

> (2001). 29. Suzuki, T. Yamayam M, Sekizawa, K., Hosada, M.,

Yamada, N.,

> Ishizuka, S., Nakayama, K., Yanai, M., Numazaki, Y., Sasaki, H.

> Bafilomycin A. K inhibits rhinovirus infection in human airway

epithelium:

> Effects on endosome and ICAM-I. Am. J. Physiol. Lung Cell. MoI.

Physiol.

> 280 (6 24-6), L1115-L1127 (2001). 30. Guinea, R. & Carrasco, L.

> Requirement for vacuolar proton- ATPase activity during entry

influenza

> virus into cells. J. ViroJ. 69 (4), 2306-2312 (1995).

>

> [0230] 31. Moriyama, Y. & Futai, M. Presence of 5-

hydroxytryptamine

> (serotonin) transport coupled with vacuolar-type H+ATPase in

> neuroscretory

> granules from bovine posterior pituitary. J. Biol. Chem. 265 (16),

> 9165-9169

> (1990).

>

> [0231] 32. Wolosker, H., de Souza, D. O. & de Meis, L. Regulation

of

> glutamate transport into synaptic vesicles by chloride and proton

> gradient. Am.

> Soc. Biochem. MoI. Bio. 271 (20), 11726-11731 (1996). 33.Xu, C J.

et al.

> Phosphocreatine-dependent glutamate uptake by synaptic vesicles. A

> comparison

> with atp-dependent glutamate uptake. J. Bio. Chem. 271 (23), 13435-

13440

> (1996).

>

> [0232] 34. Moriyama, Y., Tsai, H. L. & Futai, M. Energy-dependent

> accumulation of neuron blockers causes selective inhibition of

> neurotransmitter

> uptake by brain synaptic vesicles. Arch. Biochem. Biophys. 305 (2),

> 278-281

> (1993).

>

> [0233] 35. Moriyama Y. & Futai, M. Presence of 5-hydroxytryptamine

> (serotonin)

> transport coupled with vacuolar-type H (+) -ATPase in

neurosecretory

> granules

> from bovine posterior pituitary. J. Biol. Chem. 265 (16), 9165-9169

> (1990).

>

> [0234] 36. Wolosker, H., de Souza, D. O. & de Meis, L. Regulation

of

> glutamate transport into synaptic vesicles by chloride and proton

> gradient. J.

> Biol. Chem. 271 (20), 11726-11731 (1996). 37. Brochier, G. &

Morel, N.

> The

> same 15 kDa proteolipid subunit is a constituent of two different

> proteins in

> torpedo, the acetylcholine releasing protein mediatophore and the

> vacuolar

> H+ATPase. Neurochem. Int. 23 (6), 525-539 (1993).

>

> [0235] 38. Hayashi, M., Haga, M., Yatsushiro, S., Yamamoto, A. &

> Moriyama,

> Y. Vesicular monoamine transporter 1 is responsible for storage of

>

> [0236] 5-hydroxytryptamine in rat pinealocytes. J. Neurochem. 73

(6),

> 2538-2545 (1999).

>

> [0237] 39. Terland, O. & Flatmark, T. Drug-induced parkinsonism:

> cinnarizine

> and flunarizine are potent uncouplers of the vacuolar H+-ATPase in

> catecholamine

> storage vesicles. Neuropharmacology 38 (6), 879-882 (1999).

>

> [0238] 39. Miyamoto, J. K., Uezu, E., Jiang, P. J. & Miyamoto, A.

T. H

> (+) - ATPase and transport of DOPAC, HVA, and 5-HIAA in monoamine

> neurons.

> Physiol. Behav. 53 (1), 65-74 (1993). 40. Ramsay, A. M. & Rundle,

A.

> Clinical and biochemical findings in ten patients with benign

rayalgic

> encephalomyelitis. Postgrad. Med. J. 55 (654), 856-857 (1979).

>

> [0239] 41. Condon BR, Chaudhuri A, Condon BR, Gow JW, Hadley DN,

Brennan

> D,

> " Proton magnetic resonance spectroscopy of basal ganglia in chronic

> fatigue

> syndrome " Neuroreport 14 (2), 225-228 (2003)

>

> [0240] 42. Chaudhuri A, Gow JW Behan PO, " Chronic fatigue syndrome

and

> systemic

> viral infections: current evidence and recent advances. " in " Brain

> Disease -

> Therapeutic Strategies and Repair " Abramsky 0, Compston DA,

A,

> Said G.

> 2002 pp 127-135.

>

> [0241] 43. Vernon, SD, Unger, ER, Dimulescu, IM, Rajeevan, M and

Reeves,

> WC.

> Utility of the blood for gene expression profiling and biomarker

> discovery in

> chronic fatigue syndrome. Disease Markers 18, 193-199 (2002). 44.

Klimas

> NG,

> Salvato FR, R, Fletcher MA. Immunological abnormalities in

chronic

> fatigue syndrome. J Clin Microbiol 1990; 28: 1403-10.

>

> [0242] 45. Lloyd AR, Wakefield D, Hickie I. Immunity and the

> pathophysiology of

> chronic fatigue syndrome. Ciba Found Symp 1993; 173: 176-87.

>

> [0243] 46. Barker E, Fuj imara SF, Fadem MB, Landay AL, Levy JA.

> Immunological

> abnormalities associated with chronic fatigue syndrome, Clin

Infect Dis

> 1994;

> 18 (Suppl 1): S136-41.

>

> [0244] 47. Hassan IS, Bannister BA, Akbar A, Weir W, Bofill M. A

study of

> the

> immunology of the chronic fatigue syndrome: correlation of

immunologic

> parameters to health dysfunction. Clin Immunol Immunopathol 1998;

87:

> 60-7.

>

> [0245] 48. Vollmer-Conna U, Lloyd A, Hickie I, Wakefield D. Chronic

> fatigue

> syndrome: an immunological perspective. Aust NZ Psychiatry 1998;

32:

> 523-7.

> Table 6

>

> [0246]

> <EMI ID=52.1>

>

> [0247]

> <EMI ID=52.2>

>

> [0248]

> <EMI ID=53.1>

>

> [0249]

> <EMI ID=54.1>

>

> [0250]

> <EMI ID=55.1>

>

> [0251]

> <EMI ID=56.1>

>

> [0252]

> <EMI ID=57.1>

>

> [0253]

> <EMI ID=58.1>

>

> [0254]

> <EMI ID=59.1>

>

> [0255]

> <EMI ID=60.1>

>

> [0256]

> <EMI ID=61.1>

>

> [0257]

> <EMI ID=62.1>

>

> [0258]

> <EMI ID=63.1>

>

> [0259]

> <EMI ID=64.1>

>

> [0260]

> <EMI ID=65.1>

>

> [0261]

> <EMI ID=66.1>

>

> [0262]

> <EMI ID=67.1>

>

> [0263]

> <EMI ID=68.1>

>

> [0264]

> <EMI ID=69.1>

>

> [0265]

> <EMI ID=70.1>

>

> [0266]

> <EMI ID=71.1>

>

> [0267]

> <EMI ID=72.1>

>

> [0268]

> <EMI ID=73.1>

>

> [0269]

> <EMI ID=74.1>

>

> [0270]

> <EMI ID=75.1>

>

> [0271]

> <EMI ID=76.1>

>

> [0272]

> <EMI ID=77.1>

>

> [0273]

> <EMI ID=78.1>

>

> [0274]

> <EMI ID=79.1>

>

> [0275]

> <EMI ID=80.1>

>

> [0276]

> <EMI ID=81.1>

>

> [0277]

> <EMI ID=82.1>

>

> [0278]

> <EMI ID=83.1>

>

> [0279]

> <EMI ID=84.1>

>

> [0280]

> <EMI ID=85.1>

>

> [0281]

> <EMI ID=86.1>

>

> [0282]

> <EMI ID=87.1>

>

> [0283]

> <EMI ID=88.1>

>

> [0284]

> <EMI ID=89.1>

>

> [0285]

> <EMI ID=90.1>

>

> [0286]

> <EMI ID=91.1>

>

> [0287]

> <EMI ID=92.1>

>

> [0288]

> <EMI ID=93.1>

>

> [0289]

> <EMI ID=94.1>

>

> [0290]

> <EMI ID=95.1>

>

> [0291]

> <EMI ID=96.1>

>

> [0292]

> <EMI ID=97.1>

>

> [0293]

> <EMI ID=98.1>

>

> [0294]

> <EMI ID=99.1>

>

> [0295]

> <EMI ID=100.1>

>

> [0296]

> <EMI ID=101.1>

>

> [0297]

> <EMI ID=102.1>

>

> [0298]

> <EMI ID=103.1>

>

> [0299]

> <EMI ID=104.1>

>

> [0300]

> <EMI ID=105.1>

>

> [0301]

> <EMI ID=106.1>

>

> [0302]

> <EMI ID=107.1>

>

> [0303]

> <EMI ID=108.1>

>

> [0304]

> <EMI ID=109.1>

>

> [0305]

> <EMI ID=110.1>

>

> [0306]

> <EMI ID=111.1>

>

> [0307]

> <EMI ID=112.1>

>

> [0308]

> <EMI ID=113.1>

>

> [0309]

> <EMI ID=114.1>

>

> [0310]

> <EMI ID=115.1>

>

> [0311]

> <EMI ID=116.1>

>

> [0312]

> <EMI ID=117.1>

>

> [0313]

> <EMI ID=118.1>

>

> [0314]

> <EMI ID=119.1>

>

> [0315]

> <EMI ID=120.1>

>

> [0316]

> <EMI ID=121.1>

>

> [0317]

> <EMI ID=122.1>

>

> [0318]

> <EMI ID=123.1>

>

> [0319]

> <EMI ID=124.1>

>

> [0320]

> <EMI ID=125.1>

>

> [0321]

> <EMI ID=126.1>

>

> [0322]

> <EMI ID=127.1>

>

> [0323]

> <EMI ID=128.1>

>

> [0324]

> <EMI ID=129.1>

>

> [0325]

> <EMI ID=130.1>

>

> [0326]

> <EMI ID=131.1>

>

> [0327]

> <EMI ID=132.1>

>

> [0328]

> <EMI ID=133.1>

>

> [0329]

> <EMI ID=134.1>

>

> [0330]

> <EMI ID=135.1>

>

> [0331]

> <EMI ID=136.1>

>

> [0332]

> <EMI ID=137.1>

>

> [0333]

> <EMI ID=138.1>

>

> [0334]

> <EMI ID=139.1>

>

> [0335]

> <EMI ID=140.1>

>

> [0336]

> <EMI ID=141.1>

>

> [0337]

> <EMI ID=142.1>

>

> [0338]

> <EMI ID=143.1>

>

> [0339]

> <EMI ID=144.1>

>

> [0340]

> <EMI ID=145.1>

>

> [0341]

> <EMI ID=146.1>

>

> [0342]

> <EMI ID=147.1>

>

> [0343]

> <EMI ID=148.1>

>

>

> Claims of WO2006082390

> ----------------------

>

> Claims

>

> 1. A method for investigating whether a test subj ect is affected

by

> chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME),

comprising

> providing a biological sample from the subj ect and determining

the level

> in the sample of one or more biomarkers for CFS/ME, wherein the

> biomarkers

> comprise an expression product of at least one gene shown in Table

1.

>

> 2. A method according to claim 1, wherein the biomarkers comprise

> expression products of a plurality of genes shown in Table 1.

>

> 3. A method according to claim 2 wherein the biomarkers comprise

> expression products of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more

> of the genes shown in Table 1.

>

> 4. A method according to any one of claims 1 to 3 wherein the

biomarkers

> comprise expression products of one or more of defensin [alpha]1,

> haemoglobin [gamma], CXCR4, tubulin beta 1 and HLA DR[beta]4 genes.

>

> 5. A method according, to claim 4 wherein the biomarkers comprise

> expression products of two, three, four or five of defensin [alpha]

1,

> haemoglobin [gamma], CXCR4, tubulin beta 1 and HLA DR[beta]4 genes.

>

> 6. A method according to claim 4 or claim 5 wherein the biomarkers

> comprise expression products of further hub genes of Table IA.

>

> 7. A method according to any one of claims 4 to 6 wherein the

biomarkers

> further comprise expression products of one or more of

thrombospondin 1,

> caspase 1 and/or IgG3.

>

> 8. A method according to any one of claims 1 to 7 wherein the

biomarkers

> comprise expression products of at least one gene from each of at

least

> two of Tables 2, 3, 4 and 5.

>

> 9. A method according to claim 8 wherein said at least one gene

from each

> said Table is a hub gene.

>

> 10. A method according to any one of the preceding claims wherein

the

> biomarkers comprise expression products of defensin cxl, CXCR4 and

> lactotransferrin (LTF) genes.

>

> 11. A method according to claim 10 wherein the biomarkers comprise

> expression products of defensin [alpha]4, integrin [alpha]2B,

integrin

> [beta]3, arginase 1, arginase 2, thrombospondin 1, membrane

associated

> protein 17 (MAP 17 ), Charcot Leyden Crystal Protein (CLC) and/or

> chondroitin sulphate proteoglycan 2 (versican) genes.

>

> 12. A method according to any one of the preceding claims wherein

the

> biomarkers comprise an expression product of haemoglobin [gamma]

(foetal

> haemoglobin).

>

> 13. A method according to claim 12 wherein the biomarkers comprise

> expression products of haemoglobin alpha 1, haemoglobin beta,

haemoglobin

> delta, prostaglandin-endoperoxide synthase 1 and/or

> prostaglandin-endoperoxide synthase 2 genes.

>

> 14. A method according to any one of the preceding claims wherein

the

> biomarkers comprise an expression product of serine/threonine

kinase 17B

> (STK17B).

>

> 15. A method according to claim 14 wherein the biomarkers comprise

> expression products of caspase 1, dynamin 1-like, and/or

phosphatidyl

> serine binding protein genes.

>

> 16. A method according to any one of the preceding claims wherein

the

> biomarkers comprise an expression product of HLA-DR[beta]4.

>

> 17. A method according to claim 16 wherein the biomarkers comprise

> expression products of HLA-DQ[beta]l and/or immunoglobulin heavy

chain

> [gamma]3 genes.

>

> 18. A method according to any one of the preceding claims wherein

the

> biomarkers comprise an expression product of one or more further

genes

> from Table 6.

>

> 19. A method according to any one of the preceding claims wherein

the

> biomarkers comprise an expression product of prostaglandin D2

synthase.

>

> 20. A method for classifying a subject affected by CFS/ME, the

method

> comprising providing a biological sample from the subj ect and

> determining

> the level in the sample of one or more biomarkers for CFS/ME,

wherein the

> biomarkers comprise expression products of at least one gene shown

in one

> or more of Tables 2 to 5.

>

> 21. A method according to claim 20 wherein the biomarkers comprise

> expression products of a hub gene from one of Tables 2, 3, 4 or 5.

>

> 22. A method according to claim 21 wherein the biomarkers comprise

> expression products of hub genes from each of two, three or all of

Tables

> 2, 3, 4 or 5.

>

> 23. A method according to any one of claims 20 to 22 further

comprising

> determining network genes from one, two, three or all of Tables 2,

3, 4

> and 5.

>

> 24. A method according to any one of claims 20 to 23 wherein the

> biomarkers comprise expression products of defensin [alpha]1,

CXCR4 and

> lactotransferrin (LTF) genes.

>

> 25. A method according to claim 24 wherein the biomarkers comprise

> expression products of defensin [alpha]4, integrin [alpha]2B,

integrin

> [beta]3, arginase 1, arg+-nase 2, thrombospondin 1, membrane

associated

> protein 17 (MAP 17 ), Charcot Leyden Crystal Protein (CLC) and/or

> chondroitin sulphate proteoglycan 2 (versican) genes.

>

> 26. A method according to any one of claims 20 to 25 wherein the

> biomarkers comprise an expression product of haemoglobin Y (

foetal

> haemoglobin).

>

> 27. A method according to claim 26 wherein the biomarkers comprise

> expression products of haemoglobin alpha 1, haemoglobin beta,

haemoglobin

> delta, prostaglandin-endoperoxide synthase 1 and/or

> prostaglandin-endoperoxide synthase 2 genes.

>

> 28. A method according to any one of claims 20 to 27 wherein the

> biomarkers comprise an expression product of serine/threonine

kinase 17B

> (STK17B).

>

> 29. A method according to claim 28 wherein the biomarkers comprise

> expression products of caspase 1, dynamin 1-like, and/or

phosphatidyl

> serine binding protein genes.

>

> 30. A method according to any one of claims 20 to 28 wherein the

> biomarkers comprise an expression product of HLA-DR[beta]4.

>

> 31. A method according to claim 30 wherein the biomarkers comprise

> expression products of HLA-DQ[beta]l and/or immunoglobulin heavy

chain

> [gamma]3 genes.

>

> 32. A method according to any one of claims 20 to 31 comprising

> classifying the subject as being affected by one or more of

oxidative

> stress, excessive apoptosis, and immunological dysregulation (MHC

I to II

> shift).

>

> 33. A method of determining whether an individual affected by

CFS/ME is

> suitable for treatment with an anti-oxidant, minocycline or a

> corticosteroid, comprising determining whether the individual is

affected

> by one or more of oxidative stress, excessive apoptosis, and

> immunological

> dysregulation by a method according to any one of claims 20 to 24,

and

> prescribing a suitable treatment depending on the outcome.

>

> 34. A method of creating a library of expression profiles for use

in

> determining whether an individual is affected by CFS/ME, the

method

> comprising

> (a) providing biological samples from a plurality of individuals

affected

> by CFS/ME, and determining the level in each sample of one or more

> biomarkers for CFS/ME to create a plurality of expression profiles

from

> affected individuals;

> ( providing biological samples from a plurality of individuals

not

> affected by CFS/ME, and determining the level in the sample of

said one

> or

> more biomarkers for CFS/ME to create a plurality of expression

profiles

> from unaffected individuals;

> wherein the biomarkers comprise expression products of one or more

genes

> shown in Table 1.

>

> 35. A method according to claim 34 comprising the step of

retrievably

> storing each of the expression profiles on a computer data

carrier, in

> order to create a database of expression profiles for both

affected and

> unaffected individuals.

>

> 36. A method of determining the efficacy of a treatment for CFS/ME

> comprising the steps of:

> (a) providing a biological sample from a subj ect affected by

CFS/ME who

> has been subj ected to said treatment, ( determining the level

in said

> sample of one or more biomarkers for CFS/ME to create an

expression

> profile for said subj ect, and

> [c) comparing said expression profile with

> i) a comparable expression profile obtained from said test subject

before

> initiation of said treatment, and/or

> ii) a comparable expression profile obtained from said test

subject at an

> earlier stage of said treatment, and/or

> iii) a comparable expression profile characteristic of a subj ect

who is

> unaffected by CFS/ME,

> wherein the one or more biomarkers for CFS/ME comprise expression

> products

> of one or more genes shown in Tables 1 to 5.

>

> 37. A method according to any one of the preceding claims wherein

the

> biological sample is a blood sample or comprises peripheral blood

> mononuclear cells isolated from a blood sample.

>

> 38. A method according to any one of the preceding claims wherein

the

> expression level of each of the biomarkers is determined by

contacting

> the

> sample with a binding agent capable of binding specifically to

said

> expression product of the corresponding gene.

>

> 39. A method according to claim 38 wherein the expression product

is a

> nucleic acid.

>

> 40. A method according to claim 39 wherein the binding agent is a

nucleic

> acid probe or primer.

>

> 41. A method according to claim 39 or claim 40 wherein the

expression

> product is mRNA, pre-r[alpha]RNA or cDNA.

>

> 42. A method according to any one of claims 39 to 41 wherein the

> expression level of the biomarker is determined by means of

Northern

> blot,

> in situ hybridisation, RT-PCR, Southern blot or microarray assay.

>

> 43. A method according to claim 38 wherein the expression product

is a

> polypeptide.

>

> 44. A method according to claim 43 wherein the binding agent is a

ligand

> or receptor for said polypeptide, or an antibody or fragment

thereof

> specific for said polypeptide

>

> 45. A method according to claim 43 or claim 44 wherein the

expression

> level of the biomarker is determined by Western blot, ELISA or

microarray

> assay.

>

> 46. A method according to any one of the preceding claims further

> comprising the step of determining the level, in the biological

sample,

> or in a further biological sample from the subj ect, of the

peptide QYNAD.

>

> 47. A method according to claim 46 wherein the further biological

sample

> is a blood or serum sample.

>

> 48. A method according to claim 46 wherein the further biological

sample

> is cerebrospinal fluid.

>

> 49. A kit for use in a method according to any one of the preceding

> claims,

> the kit comprising a plurality of binding agents, each capable of

> binding specifically and individually to an expression product of

one of

> the genes of Table 1, or the peptide QYNAD.

>

> 50. A kit according to claim 49 comprising binding agents specific

for

> expression products of two or more genes of Tables 1, or at least

one

> gene of Table 1 and the peptide QYNAD.

>

> 51. A kit according to claim 50 comprising binding agents capable

of

> binding specifically and individually to expression products of 2,

3, 4,

> 5, 6, 7, 8, 9, 10, 15, 20 or more of the genes of Table 1 and

> optionally the peptide QYNAD.

>

> 52. A kit according to any one of claims 49 to 51 wherein the

binding

> agents are immobilised on one or more solid supports.

>

> 53. A kit according to any one of claims 49 to 52 further

comprising one

> or more binding agents capable of binding specifically to an

expression

> product of a control gene which is substantially not

differentially

> expressed between individuals affected and whose expression level

is

> substantially unaffected by CFS/ME.

>

> 54. A kit according to claim 53 wherein the control gene is

> glyceraldehyde

> phosphate dehydrogenase (GAPDH), [beta]-actin, or abl (ableson

tyrosine

> kinase).

>

> 55. A kit according to any one of claims 49 to 54 comprising

binding

> agents for expression products of less than 1000 different genes,

less

> than 500 different genes, less than 100, less than 50, less than

40,

> less than 30, less than 20, or less than 10 different genes.

>

> 56. A kit according to any one of claims 49 to 55 wherein the

expression

> product is a nucleic acid and the binding agent is a nucleic acid

probe

> or

> primer.

>

> 57. A kit according to claim 56 wherein the expression product is

mRNA,

> pre-mRNA or cDNA.

>

> 58. A kit according to any one of claims 49 to 55 wherein the

expression

> product is a polypeptide and the binding agent is a ligand or

receptor

> for

> said polypeptide, or an antibody or fragment thereof specific for

said

> polypeptide

>

> 59. An expression profile database, comprising a plurality of

expression

> profiles of biomarkers for CFS/ME from affected and unaffected

> individuals

> , wherein the biomarkers comprise expression products of one or

more

> genes

> shown in Table 1.

>

> 60. A method of treating CFS/ME in an individual suffering

therefrom,

> comprising administering an effective amount of an antioxidant.

>

> 61. A method according to claim 60 further comprising

administering

> therapeutically effective amounts of a corticosteroid and/or

minocyline.

>

> 62. Use of an anti-oxidant in the preparation of a medicament for

the

> treatment of CFS/ME.

>

> 63. Use according to claim 62 wherein the medicament is formulated

for

> administration in conjunction with a corticosteroid and/or

minocycline,

> or

> comprises a corticosteroid and/or minocycline.

>

> 64. A method according to claim 60 or claim 61, or use according

to claim

> 62 or claim 63, wherein the anti-oxidant is coenzyme Q10 or an

inhibitor

> of a cyclooxygenase (COX) enzyme such as celecoxib (4- [5-

> (4-methylphenyl) -3- (trifluoromethyl ) -1H-pyrazol-1-yl]

> benzenesulfonamide).

>

> 65. A method of treating CFS/ME in an individual suffering

therefrom,

> comprising administering an effective amount of minocycline.

>

> 66. A method according to claim 65 further comprising

administering a

> therapeutically effective amount of a corticosteroid and/or an

> anti-oxidant.

>

> 67. Use of minocycline in the preparation of a medicament for the

> treatment of CFS/ME.

>

> 68. Use according to claim 67 wherein the medicament is formulated

for

> administration in conjunction with a corticosteroid and/or an

> anti-oxidant, or comprises a corticosteroid and/or an anti-oxidant.

>

> 69. A method of treating CFS/ME in an individual suffering

therefrom,

> comprising administering an effective amount of a corticosteroid.

>

> 70. A method according to claim 69 further comprising

administering a

> therapeutically effective amount of minocycline and/or an anti-

oxidant.

>

> 71. Use of a corticosteroid in the preparation of a medicament for

the

> treatment of CFS/ME.

>

> 72. Use according to claim 71 wherein the medicament is formulated

for

> administration in conjunction with minocycline and/or an

antioxidant, or

> comprises minocycline and/or an anti-oxidant.

>

> 73. A method according to claim 69 or claim 70, or use according

to claim

> 71 or claim 72, wherein the corticosteroid is hydrocortisone,

> dexamethasone or prednisone.

>

> 74. A pharmaceutical composition comprising a therapeutically

effective

> amount of minocycline in combination with a therapeutically

effective

> amount of a corticosteroid and/or an anti- oxidant, and a

> pharmaceutically

> acceptable carrier..

>

> 75. A pharmaceutical composition comprising a therapeutically

effective

> amount of a corticosteroid in combination with a therapeutically

> effective

> amount of minocycline and/or an anti- oxidant, and a

pharmaceutically

> acceptable carrier.

>

> 76. A pharmaceutical composition comprising a therapeutically

effective

> amount of an anti-oxidant in combination with a therapeutically

effective

> amount of a corticosteroid and/or minocycline, and a

pharmaceutically

> acceptable carrier.

>

>

> Mosaics of WO2006082390

> -----------------------

>

> [see European Patent Office Document

>

> http://v3.espacenet.com/textdraw?

DB=EPODOC & IDX=WO2006082390 & F=0 & QPN=WO2006082390

>

>

> Original Document of WO2006082390

> ---------------------------------

>

> [see European Patent Office Document

>

> http://v3.espacenet.com/origdoc?

DB=EPODOC & IDX=WO2006082390 & F=0 & QPN=WO2006082390

>

>

> INPADOC legal status of WO2006082390

> ------------------------------------

>

> No legal data found.

>

> --------

> © 2006 European Patent Office

>

[Guest guest]

No, but I know that corticosteroids are an absolute no-no if you have

lyme/borrelia as it has a supressive effect on the immune system.

Is minocycline not an ABX commonly used for borrelia, arthritis etc?

And the Co Q is already being used by many with CFS over here in the

UK who have come up with mitochondrial problems via the ATP profile

test. It is one of the supplements we are put on for that.

BW,

Sheila

> >

> > Hi All,

> > This has just been posted on a couple of lists. Looks very

> interesting.

> > BW,

> > Sheila

> >

> > Source: European Patent Office

> > Date: August 10, 2006

> > URL: http://v3.espacenet.com/textdoc?

> DB=EPODOC & IDX=WO2006082390 & F=0

> > Ref: http://www.me-net.dds.nl/forward/genomics.html

> >

> >

> > Bibliographic data of WO2006082390

> > ----------------------------------

> >

> > Publication number: WO2006082390

> > Publication date: 2006-08-10

> > Inventor: GOW JOHN (GB);

> > CHAUDHURI ABHIJIT (GB)

> > Applicant: UNIV GLASGOW (GB);

> > GOW JOHN (GB);

> > CHAUDHURI ABHIJIT (GB)

> > Classification: international: C12Q1/68; G01N33/53; C12Q1/68;

> > G01N33/53;

> > european: Application number: WO2006GB00332

> 20060201

> > Priority number(s): GB20050002042 20050201

> > View INPADOC patent family

> > Cited documents: XP002361324

> > XP002377235

> > XP002377236

> > XP005062766

> > XP002377237

> >

> >

> > Abstract of WO2006082390

> > ------------------------

> >

> > The invention relates to materials and methods for diagnosis and

> > treatment of

> > chronic fatigue syndrome/myalgic encephalitis. A number of genes

> are

> > identified

> > which are expressed at abnormal levels in patients affected by

> CFS/ME as

> > compared to normal healthy individuals. These genes include those

> > encoding

> > defensin a1, haemoglobin, CXCR4, tubulin beta 1, serine/threonine

> kinase

> > 17B,

> > HLA DRss4 and prostaglandin D2 synthase. The genes identified

> provide

> > objective

> > disease markers that may be used in diagnostic tests to support the

> > diagnosis of

> > CFS/ME or for monitoring the effectiveness of therapy. They also

> provide

> > a

> > rational basis for classifying CFS/ME patients according to the

> > biochemical

> > lesion underlying their symptoms and enable provision of

> appropriate

> > targeted

> > therapies.

> >

> >

> > Description of WO2006082390

> > ---------------------------

> >

> > [0001] Materials and Methods for Diagnosis and Treatment of Chronic

> > Fatigue

> >

> > [0002] Syndrome

> >

> > [0003] Field of the Invention The invention relates to chronic

> fatigue

> > syndrome/myalgic encephalitis, and in particular to materials and

> methods

> > for

> > its diagnosis and treatment.

> >

> > [0004] Background to the Invention Chronic fatigue syndrome (CFS),

> also

> > known

> > as Myalgic

> >

> > [0005] Encephalomyelitis (ME), is an acquired disorder with long

> term

> > disability<1>. The illness affects both children and adults and is

> > characterised

> > by persistent or relapsing fatigue of sufficient severity to

> interfere

> > with

> > normal function. In addition, patients experience impairment in

> short

> > term

> > memory and concentration, muscle pain and prolonged post-exertional

> > malaise<2>.

> > The estimated overall prevalence of CFS/ME in the community is

> > approximately

> > 0.2-0.4% in adults and 0.07% in adolescents and children<3>.

> >

> > [0006] The pathogenesis of CFS/ME is unknown. Diagnosis of CFS/ME

> > according to

> > the internationally accepted definition (modified CDC criteria) is

> > essentially

> > based on exclusion of known medical and psychiatric diseases<2>.

> As yet,

> > there

> > is no specific or sensitive diagnostic test that positively

> establishes

> > and/or

> > supports the clinical diagnosis of CFS/ME.

> >

> > [0007] Nevertheless, in 2002, following the publication of a UK

> > Government

> > Working Party report, Professor Sir Liam son confirmed that

> CFS/ME

> > is a

> > debilitating and distressing condition affecting many people which

> should

> > be

> > classed alongside other diseases such as multiple sclerosis and

> motor

> > neurone

> > disease " .

> >

> > [0008] A significant proportion of patients report antecedent

> history of

> > community acquired viral or bacterial infections <4>'<5>'<6>. in

> > addition, it is

> > clear that patients with CFS/ME have a hypoactive

> > hypothalamic-pituitary-adrenal

> > (HPA) axis with altered neuroendocrine regulation affecting

> > neurotransmitters

> > such as monoamines (norepinephrine, serotonin and dopamine) and

> > acetylcholine in

> > the central nervous system<4>. Recent studies have demonstrated

> that

> > viruses can

> > affect neurotransmitter functions<7>'<8>.

> >

> > [0009] At the cellular level, fatigue has been linked with

> alterations in

> > the

> > cell membrane ion-channel traffic and ATPase system<9>. ATPases

> are also

> > linked

> > with neurotransmitter release (e. g. dopamine) <10> and cellular

> energy

> > metabolism via creatine phosphatase. Increased

> >

> > [0010] ATPase activity has been reported in muscle biopsies from

> patients

> > with

> > CFS <11>' <12>. Previous work<11>' <13> has raised the possibility

> that

> > patients

> > with CFS may have an ion channel dysfunction. This dysfunction

> might be

> > induced

> > by changes in the ion channel function, neurotransmitters involved

> in

> > " gating "

> > the channel or by a shift in the balance of the cellular " energy

> charge " ,

> > i.e. the ratio between ATP and ADP that is normally a function of

> the

> > ATPase

> > activity.

> >

> > [0011] One previous study<43> has identified a number of genes

> > differentially

> > expressed between PBMCs from CFS/ME patients and healthy controls.

> A

> > number of

> > the genes identified are implicated in various aspects of

> immunological

> > function, from which the authors concluded that some kind of

> > immunological

> > dysfunction may be involved in pathogenesis of CFS/ME. The authors

> were

> > unable

> > to relate the observed expression patterns to any functional model

> of

> > disease

> > etiology or pathology, and did not suggest that any of the genes

> which

> > they

> > identified could serve as useful biomarkers for CFS/ME. Their

> conclusions

> > are

> > consistent with previous studies which have suggested immunological

> > abnormality

> > in CFS/ME (Refs. 44 to 48) but the precise mechanism of

> immunological

> > dysfunction in CFS/ME has not been established.

> >

> > [0012] Summary of the Invention

> >

> > [0013] The present inventors have identified a number of genes

> which are

> > expressed at abnormal levels in patients affected by CFS/ME as

> compared

> > to

> > normal healthy individuals. In contrast to the earlier studies

> described

> > above,

> > the present inventors have been able to use the expression

> patterns of

> > these

> > genes to establish functional models of various aspects of the

> pathology

> > of

> > CFS/ME, which explain many of the symptoms observed in affected

> > individuals.

> > These provide a rational basis for classifying CFS/ME patients

> according

> > to the

> > biochemical lesion underlying their symptoms and enable appropriate

> > targeted

> > therapies to be provided for the first time.

> >

> > [0014] The genes identified in the present invention provide obj

> ective

> > disease

> > markers that may be used in diagnostic tests to support the

> diagnosis of

> > CFS/ME

> > or in other applications. For example, the tests may enable

> deselection

> > of

> > inappropriately diagnosed patients who have an alternative

> diagnosis for

> > their

> > fatigue symptoms. The tests may also be used to classify patients

> > according to

> > the particular biochemical basis for their symptoms. In turn, this

> may

> > enable a

> > clinician to identify therapies which would be appropriate and

> rule out

> > those

> > which would not. The tests may also be applied as an outcome

> measure for

> > interventional trials in CFS/ME, not least because no specific

> treatment

> > is

> > currently considered to be effective in the patient population

> defined by

> > the

> > clinical criteria. Further, the tests may offer support to CFS/ME

> > patients for

> > their claims for disability and insurance. Finally, depending on

> the

> > specificity and sensitivity of the test, one may apply it to screen

> > patients

> > with symptoms of chronic fatigue to identify or exclude CFS/ME.

> Given the

> > fact

> > that chronic fatigue as a symptom is ten times commoner in the

> population

> > (2%)

> > than CFS/ME, an estimated size of the market for a screening test

> is at

> > least

> > 10 million in the UK alone.

> >

> > [0015] Thus the present invention provides a method for

> investigating

> > whether a

> > test subj ect is affected by chronic fatigue syndrome/myalgic

> > encephalomyelitis

> > (CFS/ME), the method comprising providing a biological sample from

> the

> > subj ect

> > and determining the level in the sample of a biomarker for CFS/ME,

> > wherein the

> > biomarker is an expression product of a gene shown in Table 1.

> Genes in

> > Table 1

> > have been found to be overexpressed in CFS/ME compared to

> unaffected

> > individuals.

> >

> > [0016] The method may comprise determining the level, in the

> sample, of a

> > plurality of biomarkers (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10,

> 15, 20

> > or more biomarkers), wherein each of the biomarkers is an

> expression

> > product

> > of a gene shown in Table 1.

> >

> > [0017] The method may comprise the step of determining the level

> in the

> > sample

> > of one or more further biomarkers, being expression products of

> one or

> > more

> > genes from Table 6. Indeed, an expression product from any one of

> the

> > genes of

> > Table 6 may be used as a biomarker for CFS/ME, either alone or in

> > combination

> > with other genes of Table 6 or of Tables 1 to 5.

> >

> > [0018] The method may be considered to provide an expression

> profile of

> > the one

> > or more biomarkers for CFS/ME, for the test subj ect, at the time

> of

> > sampling.

> > By " expression profile " is meant a set of data relating to the

> level of

> > expression of one or more of the relevant biomarkers in a test

> subject,

> > in a

> > form which allows comparison with comparable expression profiles

> (e.g.

> > from

> > affected and/or unaffected individuals), in order to assist in the

> > determination of whether or not the subject is affected by CFS/ME.

> >

> > [0019] The method typically involves correlating the results

> obtained

> > with a

> > probability that the subj ect is affected by CFS/ME. In order to

> assist

> > with

> > this correlation, the method of the invention may comprise the

> step of

> > comparing

> > the expression profile for the test subj ect with one or

> more " reference "

> > expression profiles, that is to say one or more expression

> profiles

> > characteristic of unaffected subj ects (i.e. subj ects not

> suffering from

> > CFS/ME), and/or one or more expression profiles characteristic of

> > affected subj

> > ects (i.e. subj ects not suffering from CFS/ME). Thus the level of

> > expression of one or more of the said biomarkers in the test subj

> ect is

> > typically compared with that characteristic of affected and/or

> unaffected

> > individuals.

> >

> > [0020] The reference expression profiles may be profiles previously

> > derived from

> > healthy or affected individuals, or may be artificial profiles

> which

> > display

> > expression levels of the relevant biomarkers which are

> characteristic of

> > the

> > relevant group. For example, they may be computer-generated from a

> > plurality of

> > profiles previously derived from appropriate individuals. The

> profile (s)

> > characteristic of affected subj ects may be divided into

> subgroups, e.g.

> > according to their symptoms, as described in more detail below.

> >

> > [0021] Taken alone, the expression profile for the test subj ect

> may not

> > provide

> > an absolute diagnosis of CFS/ME. Normally, a clinician will also

> take

> > account

> > of the physical and/or psychological symptoms of the subj ect in

> order to

> > reach

> > a diagnosis. However the expression profile generated by the

> methods of

> > the

> > invention provides useful data to help the clinician confirm or

> rej ect a

> > preliminary diagnosis based on physical and psychological symptoms

> alone.

> > A

> > finding that one or more of these genes is upregulated in a

> particular

> > individual may therefore provide support for a diagnosis of CFS/ME.

> >

> > [0022] By " upregulated " or " overexpressed " is meant that the gene

> in

> > question

> > shows at least a two fold increase in expression at the level of

> > r[alpha]RNA

> > and/or protein as compared to the level observed in unaffected

> > (preferably

> > healthy) individuals. If desired, the level of overexpression

> required to

> > regard a result as positive may be set higher than this, e.g. a

> three,

> > four,

> > five, six, seven, eight, nine or ten fold increase as compared to

> > unaffected

> > individuals, or higher if required.

> >

> > [0023] The genes in Table 1 not only provide biomarkers for

> CFS/ME, they

> > also

> > provide the first evidence of specific biochemical pathways which

> may be

> > dysregulated in CFS/ME. They can be clustered into a number of

> subgroups,

> > based on their involvement in the same or related biochemical

> pathways

> > whose

> > dysregulation is likely to play a role in either the underlying

> cause or

> > the

> > symptoms of the disease state. Previously it has not been possible

> to

> > identify

> > such pathways, which is a primary reason why no generally-

> recognised

> > therapies

> > exist for CFS/ME. The subgroups of genes identified by the present

> > inventors

> > provide an explanation for many of the symptoms displayed by those

> > affected by

> > CFS/ME, suggest rational therapies for the condition, and provide

> means

> > for

> > monitoring the efficacy of any therapy administered.

> >

> > [0024] Without wishing to be bound by any particular theory, the

> > inventors

> > believe that CFS/ME is not a genetic disease caused by a single or

> > multiple gene

> > defects. Rather, they believe CFS/ME to be an acquired condition

> where

> > there is

> > a shift in the functional systems of a select number of genes

> regulating

> > specific biological functions (e.g. infection and immunity, cell

> membrane

> > function and cell cycle) Based on the expression patterns they have

> > observed,

> > they have established a model of " hub " and " network " genes which

> > emphasises the

> > interrelation of these key genes, with the hub genes being the

> control

> > centre

> > and the network genes being largely the effector arm of this

> functional

> > system.

> > This set of hub and network genes defines the functional shift in

> the

> > biological

> > systems of patients who continue to have symptoms due to CFS/ME.

> >

> > [0025] Thus the genes of Table 1 are subdivided into " hub " genes

> and

> >

> > [0026] " network " genes. Essentially, in any particular pathway the

> hub

> > gene(s)

> > can be considered to lie upstream of the network genes, in that

> > dysregulation

> > of the hub gene is likely to lead (directly or indirectly) to

> > dysregulation of

> > the downstream genes. In Table 1, the hub genes are shown in

> section A,

> > and the

> > network genes in section B.

> >

> > [0027] Certain genes which appear to be highly significant (e.g.

> based on

> > the

> > difference in their expression between the disease state and the

> normal

> > state)

> > have been allocated to the " hub " group even where no related set of

> > downstream

> > " network " genes is specifically identified.

> >

> > [0028] Exemplary groups of genes identified in Table 1 are

> detailed below.

> >

> > [0029] Defensin [alpha]1, CXCR4 and lactotransferrin (LTF) are all

> > involved in

> > the response to intracellular infection. The network genes

> associated

> > with

> > these hub genes include defensin [alpha]4, integrin [alpha]2B,

> integrin

> > [beta]3,

> > arginase 1, arginase 2, thrombospondin 1, membrane associated

> protein 17

> > (MAP 17), Charcot Leyden Crystal Protein (CLC) and chondroitin

> sulphate

> > proteoglycan 2 (versican). These genes are shown in Table 2.

> >

> > [0030] Haemoglobin Y (foetal haemoglobin) is part of the oxidative

> stress

> > response pathway, and is only expressed under conditions of

> oxidative

> > stress.

> > Network genes associated with the oxidative stress pathway include

> other

> > haemoglobin genes including haemoglobin alpha 1,

> > prostaglandin-endoperoxide

> > synthase 1 and prostaglandin- endoperoxide synthase 2. These genes

> are

> > shown in

> > Table 3. However it will be noted that Table 6 includes genes for

> > haemoglobins

> > alpha, beta, gamma and delta. Any one of the haemoglobin genes of

> Table 6

> > may

> > be used as a biomarker for CFS/ME, and may be used as well as, or

> in

> > place of

> > haemoglobin gamma or alpha if desired. For simplicity, though,

> reference

> > will be

> > made primarily to haemoglobin gamma.

> >

> > [0031] Serine/threonine kinase 17B (STK17B) is implicated in

> apoptosis,

> > as are

> > the network genes caspase 1, dynamin 1-like, and phosphatidyl

> serine

> > binding

> > protein. These genes are shown in Table 4.

> >

> > [0032] The MHC class II gene HLA-DR[beta]4 is upregulated in CFS/ME

> > implying a

> > shift from antigen presentation by MHC class I to MHC II

> presentation.

> > Associated network genes include HLA-DQ[beta]1 and the

> immunoglobulin

> > heavy

> > chain [gamma]3 (IgG3). These genes are shown in Table 5. IgG1 may

> be used

> > as a

> > bior[alpha]arker as well as, or in place of, IgG3.

> >

> > [0033] Methods for investigating whether a test subj ect is

> affected by

> > CFS/ME

> > may therefore comprise determining the level of at least one gene

> from

> > each of

> > at least two of Tables 2, 3, 4 and 5, and preferably from each of

> three

> > or

> > four of said Tables. In preferred embodiments, at least one gene

> from

> > each

> > said Table is a hub gene.

> >

> > [0034] Preferred genes of Table 1 are defensin [alpha]1,

> haemoglobin

> > [gamma],

> > CXCR4, tubulin beta 1 and HLA DR[beta]4. The method may involve

> testing

> > expression of any one, two, three, four or all five of these

> genes, and

> > optionally further hub genes of Table 1A.

> >

> > [0035] Additionally or alternatively, the method may involve

> testing

> > expression

> > of one or more of TSP 1, caspase 1 and/or IgG3.

> >

> > [0036] The method may also comprise the step of determining the

> level of

> > an

> > expression product of prostaglandin D2 synthase in sample. In

> contrast to

> > the

> > genes of Table 1, prostaglandin D2 synthase is found to be

> downregulated

> > in

> > individuals suffering from CFS/ME as compared to normal controls.

> Thus a

> > finding that prostaglandin D2 synthase is downregulated in a

> particular

> > individual may provide support for a diagnosis of CFS/ME.

> >

> > [0037] By " downregulated " or " underexpressed " is meant that

> prostaglandin

> > D2

> > synthase shows at least two fold higher expression at the level of

> mRNA

> > and/or

> > protein in unaffected (preferably healthy) individuals than is

> observed

> > in the

> > test subj ect. If desired, the level of downregulation required to

> regard

> > a

> > result as positive may require the level found in unaffected

> individuals

> > to be

> > three, four, five, six, seven, eight, nine or ten fold higher than

> in

> > affected

> > individuals. The method may further involve the step of

> determining the

> > level,

> > in a biological sample from the subj ect, of the peptide QYNAD, as

> > described in

> > more detail below. The level of the peptide in the sample may form

> part

> > of the

> > expression profile established for the subj ect. An elevated level

> of the

> > peptide as compared to unaffected individuals may be indicative of

> > CFS/ME. By

> > an " elevated " level is meant at least twice the level found in

> unaffected

> > healthy controls, and preferably at least 5 times or at least 10

> time the

> > level

> > found in unaffected healthy controls.

> >

> > [0038] It will be appreciated that individual patients who each

> satisfy

> > the

> > criteria for CFS/ME may nevertheless present a range of very

> different

> > symptoms.

> > Some of these can be explained by the groups of genes identified

> herein.

> >

> > [0039] For example, CFS/ME has previously been suggested to involve

> > immune

> > dysfunction. Some CFS/ME sufferers are particularly prone to

> infection by

> > viruses (e.g. influenza) and other pathogens; indeed some suffer

> > recurrent

> > infections. Others are affected by atopic/allergic symptoms. These

> > symptoms

> > may be associated with the apparent shift from Type I to Type II

> antigen

> > presentation by the MHC, which could impair the body' s ability to

> deal

> > efficiently with infections and could also exacerbate

> allergy/atopy.

> >

> > [0040] Some CFS/ME sufferers describe their body as feeling

> prematurely

> > aged,

> > and can display restricted mobility characteristic of much older

> > individuals.

> > This might be explained by excessive apoptosis, particularly in the

> > central

> > nervous system. Therefore patients having these symptoms might be

> > expected to

> > show increased expression of serine/threonine kinase 17B (STK17B)

> and its

> > associated network genes.

> >

> > [0041] Oxidative stress is also a cause of apoptosis, particularly

> in the

> > nervous system. Thus patients with increased expression of

> haemoglobin Y

> > and its

> > associated network genes may also show, or be at risk of,

> accelerated

> > programmed

> > cell death (increased level of apoptosis), particularly neuronal

> > apoptosis.

> > Features of oxidative stress and neuronal apoptosis may include

> ageing,

> > cognitive impairment and chronic pain.

> >

> > [0042] The genes of Table 2 are implicated in cellular protection

> > ( " defence " )

> > against viral or bacterial infections. Thus an overexpressed

> defensin

> > gene in

> > CFS/ME would be consistent with immune activation and correlate

> with

> > symptoms of

> > recurrent influenza-type symptoms, sore throat and lymph node

> enlargement

> > experienced by patients with CFS/ME.

> >

> > [0043] In a further aspect, the invention therefore provides a

> method for

> > classifying a subj ect affected by CFS/ME, the method comprising

> > providing a

> > biological sample from the subject and determining the level in the

> > sample of a

> > biomarker for CFS/ME, wherein the biomarker is an expression

> product of a

> > gene

> > shown in one or more of Tables 2 to 5.

> >

> > [0044] Preferably the biomarker is a hub gene from one of Tables

> 2, 3, 4

> > or 5.

> > More preferably, the method comprises determining the expression

> level of

> > hub

> > genes from each of two, three or all four of Tables 2, 3, 4 or 5.

> The

> > method

> > may also comprise determining network genes from one, two, three

> or all

> > four of

> > Tables 2, 3, 4 and 5.

> >

> > [0045] The individual can then be assigned to a subgroup of CFS/ME,

> > dependent on

> > which group or groups of genes are found to be upregulated. Thus,

> for

> > example,

> > the subj ect may be classified as being affected by one or more of

> > oxidative

> > stress, excessive apoptosis, and immunological dysregulation (MHC

> I to II

> > shift). The assignment step may involve comparing the expression

> profile

> > obtained from the subj ect with one or more expression profiles

> > characteristic

> > of individuals previously assigned to one or more subgroups of

> CFS/ME. It

> > will

> > be appreciated that the classification may be performed using the

> same

> > expression profile as that established for determining whether the

> subj

> > ect is

> > affected by CFS/ME.

> >

> > [0046] Individuals in which these particular pathways are

> dysregulated

> > may be

> > suitable for treatment by the methods described below. Therefore

> in a

> > further

> > aspect the invention provides a method of determining whether an

> > individual

> > affected by CFS/ME is suitable for treatment using such a therapy.

> The

> > method

> > comprises determining whether the individual is affected by one or

> more

> > of

> > oxidative stress, excessive apoptosis, and immunological

> dysregulation as

> > described above, and optionally prescribing a suitable treatment

> > depending on

> > the outcome. These treatments are described in detail below.

> >

> > [0047] Expression of individual biomarkers for CFS/ME (genes from

> Tables 1

> > to 5) may differ slightly between independent samples, leading to

> > slightly

> > different expression profiles for individual samples. However,

> these

> > particular

> > genes may provide a characteristic pattern of expression

> (expression

> > profile) in

> > an affected individual (i.e. one suffering from CFS/ME) that is

> > recognisably

> > different from that in an unaffected individual (i.e. one not

> suffering

> > from

> > CFS/ME).

> >

> > [0048] By creating a number of expression profiles from a number

> of known

> > affected and unaffected samples, it is possible to create a

> library of

> > profiles

> > for both sample types. The greater the number of expression

> profiles, the

> > easier it is to create a reliable characteristic expression profile

> > standard

> > (i.e. including statistical variation) that can be used as a

> control in a

> > diagnostic assay. Thus, a standard profile may be one that is

> devised

> > from a

> > plurality of individual expression profiles and devised within

> > statistical

> > variation to represent either the affected or unaffected profile.

> The

> > determination of the expression profile may be computerised and

> may be

> > carried

> > out within certain previously set parameters, to avoid false

> positives

> > and false

> > negatives.

> >

> > [0049] The computer may then be able to provide an expression

> profile

> > standard

> > characteristic of an affected sample and a normal sample. The

> determined

> > expression profiles may then be used to classify test samples as

> affected

> > or

> > unaffected as a way of diagnosis.

> >

> > [0050] Thus, in a further aspect the invention provides a method of

> > creating a

> > library of expression profiles for use in determining whether an

> > individual is

> > affected by CFS/ME, the method comprising

> >

> > [0051] (a) providing biological samples from a plurality of

> individuals

> > affected

> > by CFS/ME, and determining the level in each sample of one or more

> > biomarkers

> > for CFS/ME to create a plurality of expression profiles from

> affected

> > individuals;

> >

> > [0052] ( providing biological samples from a plurality of

> individuals

> > not

> > affected by CFS/ME, and determining the level in the sample of

> said one

> > or more

> > biomarkers for CFS/ME to create a plurality of expression profiles

> from

> > unaffected individuals;

> >

> > [0053] wherein the biomarkers comprise expression products of one

> or more

> > genes

> > shown in Table 1.

> >

> > [0054] Typically, the method comprises the step of retrievably

> storing

> > each of

> > the expression profiles on a computer data carrier, in order to

> create a

> > database of expression profiles for both affected and unaffected

> > individuals.

> >

> > [0055] The invention further provides an expression profile

> database,

> > comprising

> > a plurality of expression profiles of biomarkers for CFS/ME from

> affected

> > and

> > unaffected individuals, wherein the biomarkers comprise expression

> > products of

> > one or more genes shown in Table 1.

> >

> > [0056] It will be appreciated that the profiles may be classified

> > according to

> > their expression levels of the various groups of genes shown in

> Tables 2

> > to 5 as

> > already described. The combinations of biomarkers analysed in

> > establishing the

> > expression profiles may therefore be chosen as described above.

> >

> > [0057] The expression profiles may comprise data relating to the

> level,

> > in

> > biological samples from the subj ects, of the peptide QYNAD. If

> required,

> > the

> > methods may therefore comprise the step of determining the level

> of this

> > peptide

> > in biological samples from the subjects.

> >

> > [0058] At present there are no generally acknowledged treatments

> for

> > CFS/ME.

> > One problem which has faced researchers attempting to develop and

> > evaluate

> > suitable therapies is the lack of objective testable criteria to

> > determine their

> > efficacy. The present invention provides a useful tool for

> monitoring the

> > efficacy of an interventional treatment for CFS/ME, by studying the

> > effect of

> > the therapy, over time, on an individual' s expression profile of

> the

> > biomarkers

> > described in Tables 1 to 5. The methods described may therefore be

> used

> > to

> > evaluate the effectiveness of a test treatment, e.g. by testing it

> on a

> > population of subjects affected by CFS/ME, or to investigate an

> > individual' s

> > response to a particular therapy, to see whether or not they are

> > responding

> > appropriately.

> >

> > [0059] Thus in a further aspect the invention provides a method of

> > determining

> > the efficacy of a treatment for CFS/ME comprising the steps of:

> >

> > [0060] (a) providing a biological sample from a subj ect affected

> by

> > CFS/ME who

> > has been subj ected to said treatment, ( determining the level

> in said

> > sample

> > of one or more biomarkers for CFS/ME to create an expression

> profile for

> > said

> > subj ect, and

> >

> > [0061] © comparing said expression profile with

> >

> > [0062] i) a comparable expression profile obtained from said test

> subj

> > ect

> > before initiation of said treatment, and/or

> >

> > [0063] ii) a comparable expression profile obtained from said test

> subj

> > ect at

> > an earlier stage of said treatment, and/or

> >

> > [0064] iii) a comparable expression profile characteristic of a

> subject

> > who is

> > unaffected by CFS/ME,

> >

> > [0065] wherein the one or more biomarkers for CFS/ME are expression

> > products of

> > one or more genes shown in Tables 1 to 5. Preferred combinations of

> > biomarkers

> > have already been described in relation to earlier aspects of the

> > invention.

> >

> > [0066] In general, a treatment may be considered to be effective

> if the

> > subj

> > ect' s expression profile after treatment shows that the

> expression level

> > of one

> > or more of the biomarkers is reduced compared to its level before

> > treatment, or

> > its level earlier in a course of the treatment. Preferably the

> subj ect'

> > s

> > expression profile after treatment approaches a profile

> characteristic of

> > an

> > individual unaffected by CFS/ME. That is to say, the expression

> level of

> > each

> > of the biomarkers falls within the normal range found in unaffected

> > individuals.

> >

> > [0067] Some treatments may be targeted to particular pathways

> known to be

> > dysregulated in CFS/ME. In such cases, it may be desirable to

> follow the

> > effect of the treatment on the expression levels of genes in the

> pathway

> > likely

> > to be modulated by that treatment. For example, a treatment which

> is

> > intended

> > to reduce oxidative stress, apoptosis or immune dysfunction

> > (characterised by

> > MHC shift) may have a particular effect on the expression levels

> of the

> > genes in

> > Tables 3, 4 and 5 respectively. The biomarkers chosen to study the

> > effects of

> > the treatment may be chosen accordingly. Preferred combinations of

> > biomarkers

> > from these Tables have already been described.

> >

> > [0068] The method may further comprise determining the effect of

> the

> > treatment

> > on the level of the peptide QYNAD in a biological sample from the

> test

> > subject.

> >

> > [0069] In the diagnostic and analytical methods described herein,

> the

> > expression

> > levels of the chosen biomarker (s) for CFS/ME are preferably

> determined

> > using

> > peripheral blood mononuclear cells (PBMCs) from the test subj ect.

> > Therefore

> > the biological sample used to establish the expression profile is

> > preferably a

> > blood sample or cells isolated from a blood sample. If desired, the

> > method may

> > comprise the steps of enriching PBMCs in the sample, or isolating

> PBMCs

> > from the

> > sample.

> >

> > [0070] It will be understood that the methods described are

> generally

> > performed

> > on a biological sample which has been isolated from the test

> subject. The

> > method may, but need not, comprise the actual step of isolating the

> > sample from

> > the test subj ect, e.g. by taking a blood sample.

> >

> > [0071] Typically, the expression level of each of the biomarkers is

> > determined

> > by contacting the sample with a binding agent capable of binding

> > specifically to

> > an expression product of the genes encoding that biomarker (a gene

> of

> > Table 1, 2,

> > 3, 4, 5 or 6). Binding between the agent and an expression product

> > present

> > in the sample is then determined. If it is desired to determine the

> > expression

> > level of more than one gene, then the sample may be contacted with

> a

> > plurality

> > of binding agents, either simultaneously or sequentially, each

> binding

> > agent

> > being capable of binding specifically and individually to an

> expression

> > product

> > of one of the biomarker genes. Binding agents capable of binding to

> > nucleic

> > acid expression products (e.g. mRNA, pre-mRNA) are typically

> nucleic acid

> > primers or probes having a sequence of, or complementary to, a

> portion of

> > the

> > nucleic acid expression product. This enables the binding agent to

> > hybridise

> > under suitable conditions with the nucleic acid expression product

> > itself, e.g.

> > in a Northern blot or in situ hybridisation assay, or to a cDNA

> copy of

> > the

> > nucleic acid expression product, e.g. in a RT-PCR assay, Southern

> blot or

> > microarray assay.

> >

> > [0072] Binding agents capable of binding to polypeptide expression

> > products

> > include ligands and receptors for the polypeptide in question.

> > Particularly

> > preferred examples of binding agents are antibodies specific for

> (e.g.

> > raised

> > against) the desired polypeptide, or fragments thereof comprising

> an

> > antigen

> > binding site. These may be used in a variety of immunological assay

> > methods,

> > including Western blots and ELISA assays, as well as in microarray

> assays.

> >

> > [0073] It may be desirable to isolate expression products or

> particular

> > fractions thereof (e.g. total RNA, mRNA, total protein, soluble

> proteins

> > or

> > membrane proteins) from the sample before contacting them with the

> > binding agent.

> > The extent to which this is necessary will vary depending on the

> chosen

> > assay

> > method.

> >

> > [0074] Simple immunological assays (such as ELISA assays) or PCR-

> based

> > assays

> > using ten biomarkers or less (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10

> > biomarkers) are particularly preferred, because they can readily be

> > adapted to

> > analyse large numbers of samples in a relatively short space of

> time, at

> > relatively low cost. Such assays are also well- suited to

> automation.

> > Preferred

> > markers and combinations of markers are as described elsewhere in

> this

> > specification.

> >

> > [0075] The diagnostic and analytical methods described above may

> also

> > comprise

> > determining the level, in a biological sample from the subject, of

> the

> > peptide

> > QYNAD. It is believed that the peptide may be found in serum and

> > cerebrospinal

> > fluid (CSF) in most or all of the population, but it is found at

> elevated

> > levels

> > in subjects suffering from inflammatory and immunological

> disorders of

> > the

> > nervous system (such as multiple sclerosis and Guillain-Barre

> syndrome).

> > The

> > present inventors have now established that it is also found at

> > significantly

> > elevated levels in subj ects suffering from CFS/ME.

> >

> > [0076] The biological sample used for determining the peptide

> level may

> > be a

> > blood sample (or serum derived therefrom). Thus, conveniently, the

> same

> > blood

> > sample may be used for determining levels of the peptide and the

> other

> > biomarkers of CFS/ME. Alternatively, a different sample may be

> used, in

> > particular, a sample of cerebrospinal fluid (CSF) where available.

> >

> > [0077] The level of the peptide may be determined as described by

> > Brinkmeier et

> > al.<16>, e.g. by gel filtration chromatography. Alternatively

> > conventional

> > immunoassays such as ELISAs or Western blots may be used, employing

> > antibodies

> > raised against the peptide.

> >

> > [0078] The assay for the peptide is generally performed on a sample

> > isolated

> > from the test subj ect. The method of the invention need not

> comprise the

> > step

> > of actually isolating the sample from the test subj ect.

> >

> > [0079] In a further aspect, the invention provides a kit for use

> in a

> > diagnostic

> > or analytical method as described herein, the kit comprising a

> plurality

> > of

> > binding agents, each capable of binding specifically and

> individually to

> > an

> > expression product of one of the genes of Table I1 or the peptide

> QYNAD.

> > Thus

> > the kit includes binding agents specific for expression products

> of two

> > or more

> > genes of Tables 1, or at least one of the genes of Table 1 and the

> > peptide QYNAD.

> > The binding agents provided in the kit may be capable of binding

> > specifically

> > and individually to expression products of 2, 3, 4, 5, 6, 7, 8, 9,

> 10,

> > 15,

> > 20 or more of the genes of Table 1 and optionally the peptide

> QYNAD.

> >

> > [0080] The kit is suitable for use in the methods of the invention

> > described in

> > this specification, and may comprise instructions for performing

> one or

> > more

> > methods of the invention.

> >

> > [0081] The binding agents may be immobilised on one or more solid

> > supports.

> > Discrete supports may each carry only one type of binding agent.

> For

> > example,

> > distinct populations of beads may each carry one type of binding

> agent.

> > Alternatively, a single support may carry more than one type of

> binding

> > agent.

> > Indeed, one support (e.g. a microarray chip) may carry all of the

> > different

> > types of binding agent provided with the kit.

> >

> > [0082] In addition, the kit may comprise one or more binding agents

> > capable of

> > binding specifically to an expression product of a control gene

> which is

> > not

> > differentially expressed between individuals affected and

> unaffected by

> > CFS/ME.

> > The level of expression from this control gene may be measured m

> order to

> > assist

> > in quantification of the expression products of the genes of Table

> 1,

> > and/or

> > for quality assurance of an assay performed using the kit.

> Preferably a

> > control

> > gene is chosen which is constitutively expressed in the cells of

> the

> > biological

> > sample (i.e. always expressed, at substantially the same level,

> under

> > substantially all conditions). Such genes are often referred to as

> > " housekeeping " genes. Examples include glyceraldehyde phosphate

> > dehydrogenase

> > (GAPDH), [beta]-actin, and abl (ableson tyrosine kinase).

> >

> > [0083] The kit may comprise yet further binding agents capable of

> binding

> > to

> > expression products of other biomarker genes or control genes.

> However, m

> > preferred embodiments, the kit comprises binding agents for

> expression

> > products

> > of less than 1000 different genes, e.g. less than 500 different

> genes,

> > less

> > than 100, less than 50, less than 40, less than 30, less than 20,

> or less

> > than 10 different genes. As explained above, the groups of genes

> > identified by

> > the present inventors also suggest specific therapies for CFS/ME.

> >

> > [0084] Patients displaying signs of oxidative stress may be

> treated with

> > an

> > anti-oxidant. Thus the invention provides a method of treating

> CFS/ME in

> > an

> > individual suffering therefrom, comprising administering an

> effective

> > amount of

> > an anti-oxidant.

> >

> > [0085] Therapeutically effective amounts of a corticosteroid and/or

> > minocyline

> > may also be administered to the subject in conjunction with the

> > anti-oxidant.

> > As will be appreciated, the individual active agents may be

> administered

> > individually (in two or more separate compositions) or together

> (in the

> > same

> > composition).

> >

> > [0086] The invention further provides the use of an anti-oxidant

> in the

> > preparation of a medicament for the treatment of CFS/ME. The

> medicament

> > may be

> > formulated for administration in conjunction with a corticosteroid

> and/or

> > minocycline. Alternatively, the medicament may comprise a

> corticosteroid

> > and/or

> > minocycline.

> >

> > [0087] Examples of suitable anti-oxidants include coenzyme Q10 and

> > inhibitors of

> > cyclooxygenase (COX) enzymes, particularly COX II enzymes, such as

> > celecoxib (

> > 4- [5- (4-methyl[rho]henyl) -3- (trifluoromethyl) - 1H-pyrazol-1-

> yl]

> > benzenesulfonamide).

> >

> > [0088] Patients showing evidence of abnormal apoptosis may be

> treated

> > with

> > minocyline, which is an inhibitor of caspase 1, shown here to be

> > upregulated in

> > individuals affected by CFS/ME. Minocyline has previously been

> suggested

> > for

> > treatment of various neurological disorders including stroke,

> multiple

> > sclerosis,

> > spinal cord injury, Parkinson' s disease, Huntington' s disease

> and

> > amylotrophic lateral sclerosis - see Wee Young et al., Lancet

> Neurology,

> > 2004,

> > 744-751 for review. Thus the invention provides a method of

> treating

> > CFS/ME

> > in an individual suffering therefrom, comprising administering an

> > effective

> > amount of minocycline.

> >

> > [0089] Therapeutically effective amounts of a corticosteroid

> and/or an

> > anti-oxidant may also be administered to the subject in

> conjunction with

> > the

> > minocycline. As will be appreciated, the individual active agents

> may be

> > administered individually (in two or more separate compositions) or

> > together

> > (in the same composition).

> >

> > [0090] The invention further provides the use of minocycline in the

> > preparation

> > of a medicament for the treatment of CFS/ME. The medicament may be

> > formulated

> > for administration in conjunction with a corticosteroid and/or an

> > anti-oxidant.

> > Alternatively, the medicament may comprise a corticosteroid and/or

> an

> > anti-oxidant.

> >

> > [0091] Oxidative stress often gives rise to apoptosis. Therefore

> it may

> > be

> > advisable to treat patients showing signs of oxidative stress with

> an

> > apoptosis

> > treatment such as minocycline as a precautionary measure if they

> do not

> > already

> > show upregulation of genes involved in apoptosis.

> >

> > [0092] Patients showing signs of immune dysfunction, and

> particularly the

> > Type I

> > to II shift of MHC expression, may benefit from treatment with a

> > corticosteroid.

> > This would address the hypocortisolism previously reported in some

> CFS/ME

> > patients.

> >

> > [0093] Thus the invention provides a method of treating CFS/ME in

> an

> > individual

> > suffering therefrom, comprising administering an effective amount

> of a

> > corticosteroid.

> >

> > [0094] Therapeutically effective amounts of minocycline and/or an

> > antioxidant

> > may also be administered to the subject in conjunction with the

> > corticosteroid.

> > As will be appreciated, the individual active agents may be

> administered

> > individually (in two or more separate compositions) or together

> (in the

> > same

> > composition).

> >

> > [0095] The invention further provides the use of a corticosteroid

> in the

> > preparation of a medicament for the treatment of CFS/ME. The

> medicament

> > may be

> > formulated for administration in conjunction with minocycline

> and/or an

> > anti-oxidant. Alternatively, the medicament may comprise

> minocycline

> > and/or an

> > anti-oxidant.

> >

> > [0096] An example of a suitable corticosteroid is hydrocortisone.

> Others

> > include dexamethasone and prednisone.

> >

> > [0097] The invention further provides pharmaceutical compositions

> > suitable for

> > the treatment of CFS/ME. Thus there is provided a pharmaceutical

> > composition

> > comprising a therapeutically effective amount of minocycline in

> > combination with

> > a therapeutically effective amount of a corticosteroid and/or an

> > anti-oxidant

> > and a pharmaceutically acceptable carrier.

> >

> > [0098] Also provided is a pharmaceutical composition comprising a

> > therapeutically effective amount of a corticosteroid in

> combination with

> > a

> > therapeutically effective amount of minocycline and/or an anti-

> oxidant,

> > and a

> > pharmaceutically acceptable carrier.

> >

> > [0099] Also provided is a pharmaceutical composition comprising a

> > therapeutically effective amount of an anti-oxidant in combination

> with a

> > therapeutically effective amount of a corticosteroid and/or

> minocycline,

> > and a

> > pharmaceutically acceptable carrier.

> >

> > [0100] As described above, a particular treatment may be

> determined for

> > any

> > individual based on their expression profile of CFS/ME biomarkers.

> For

> > example,

> > an individual showing upregulation of oxidative stress pathway

> genes may

> > be

> > particularly suitable for treatment with an anti-oxidant. However

> it may

> > not be

> > convenient or necessary to establish a suitable expression profile

> before

> > beginning treatment. Therefore it may be desirable to administer

> one, two

> > or

> > all three of the treatment types described above to an individual

> > affected by

> > CFS/ME without recourse to expression profiling.

> >

> > [0101] Description of the Drawings

> >

> > [0102] Figure 1 shows a RT-PCR assay of v-ATPase mRNA in PBMCs

> from three

> > healthy control individuals (lanes 2 to 4) and three patients with

> CFS/ME

> > (lanes 5 to 7). Lane 1 contains molecular weight markers.

> >

> > [0103] Figure 2 shows a RT-PCR assay of Defensin alpha 1 mRNA in

> PBMCs

> > from

> > three healthy control individuals (lanes 2 to 4) and three

> patients with

> > CFS/ME

> > (lanes 6 to 8). Lanes 1 and 9 contain molecular weight markers.

> >

> > [0104] Figures 3 and 4 show Western blots of proteins extracted

> from

> > PBMCs from

> > healthy individuals and individuals suffering from CFS/ME, stained

> with

> > antibodies against Defensin alpha 1 and Thrombospondin 1

> respectively. In

> > each

> > case, lanes 1 and 12 contain molecular weight markers, lanes 2 to

> 11

> > contain

> > samples from patients with CFS/ME, and lanes 13 to 22 contain

> samples

> > from

> > healthy control individuals. Purified protein extract from 2x10<5>

> cells

> > was

> > loaded per lane. Proteins were separated on 10% SDS-PAGE gels and

> blotted

> > onto

> > a nylon membrane.

> >

> > [0105] Figure 5 shows a SDS-PAGE gel, stained with Coomassie blue,

> of the

> > same

> > samples used to prepare the Western blots shown in Figures 3 and

> 4. Lanes

> > 11 and

> > 22 contain molecular weight markers, lanes 1 to 10 contain samples

> from

> > patients with CFS/ME, and lanes 12 to 21 contain samples from

> healthy

> > control

> > individuals. As in Figures 3 and 4, protein extract from 2x10<5>

> cells

> > was

> > loaded per lane.

> >

> > [0106] Detailed Description of the Invention

> >

> > [0107] Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

> >

> > [0108] CFS is typically diagnosed using the modified CDC criteria

> > described by

> > Fukuda et al.<2>. All other conditions or diseases which could

> explain a

> > patient' s symptoms are first excluded. Having done this, CFS/ME is

> > diagnosed

> > if the patient has been affected by 6 months or longer of

> persistent

> > relapsing

> > or persistent fatigue accompanied by four or more concurrent

> symptoms

> > including

> > impaired memory severe enough to affect normal daily function, sore

> > throat,

> > tender lymph nodes, muscular or joint pain, new headaches,

> unrefreshing

> > sleep

> > and post-exertional malaise lasting for more than 24 hours. For the

> > purposes of

> > this specification, individuals satisfying these criteria are

> considered

> > to be

> > affected by CFS/ME.

> >

> > [0109] Assay methods

> >

> > [0110] The genes identified in Table 1 provide biomarkers which

> may be

> > used in

> > diagnostic assays to support, confirm, or refute a diagnosis of

> CFS/ME.

> > With

> > the knowledge of this set of genes, it is possible to devise many

> methods

> > for

> > determining a suitable expression profile of one or more CFS/ME

> > biomarkers in a

> > particular test sample.

> >

> > [0111] Typically, the method involves contacting expression

> products from

> > the

> > sample with a binding agent capable of binding to an expression

> product

> > of a

> > gene identified in Table 1. The expression product may be a

> transcribed

> > nucleic

> > acid sequence or an expressed polypeptide.

> >

> > [0112] The transcribed nucleic acid sequence may be mRNA or pre-

> mRNA.

> > Alternatively, the expression product may also be cDNA produced

> from said

> > mRNA.

> > The binding member may a nucleic acid having a sequence

> complementary to

> > that of

> > the RNA or cDNA which is consequently capable of specifically

> binding to

> > the

> > transcribed nucleic acid or cDNA under suitable hybridisation

> conditions,

> > e.g.

> > by Northern blotting, in situ hybridisation, or Southern blotting.

> >

> > [0113] Such protocols may use probes of at least about 20-80 bases

> in

> > length.

> > The probes may be of 100, 200, 300, 400 or 500 bases in length or

> more.

> > Binding assays may be conducted using standard procedures, such as

> > described in

> > Sambrook et al., Molecular Cloning A Laboratory Manual (New York:

> Cold

> > Spring

> > Harbor Laboratory Press, 1989 or later editions).

> >

> > [0114] RT-PCR procedures (including quantitative PCR procedures)

> may also

> > be

> > used to analyse the presence or amount of mRNA or precursor mRNA

> in a

> > given

> > sample. A suitable primer having at least 15 to 20 bases

> complementary to

> > the

> > desired mRNA or precursor mRNA sequence will typically be used to

> prime

> > cDNA

> > synthesis. Alternatively a poly-T primer (optionally comprising

> one or

> > more

> > random nucleotides at the 3' end) may be used to prime cDNA

> synthesis

> > from all

> > mRNA in the sample.

> >

> > [0115] Subsequently, a segment of the cDNA is amplified in a PCR

> reaction

> > using

> > a pair of nucleic acid primers, each typically having at least 15

> to 20

> > bases

> > complementary to the desired RNA sequence. The skilled person will

> be

> > able to

> > design suitable probes or primers based on the publicly available

> > sequence data

> > for the genes in question (see Table 1 for suitable accession

> numbers).

> >

> > [0116] Where the expression product is the expressed polypeptide,

> the

> > binding

> > member is preferably an antibody raised against or otherwise

> specific for

> > the

> > desired polypeptide, or any other molecule comprising the antigen

> binding

> > site

> > from such an antibody.

> >

> > [0117] The skilled person will realise that other binding agents

> may be

> > used as

> > appropriate. Suitable agents may include naturally- occurring

> ligands and

> > receptors for the desired polypeptide, aptamers, etc. For example,

> > aptamers

> > are nucleic acid molecules (typically DNA or RNA), selected from

> > libraries on

> > the basis of their ability to bind other molecules. Aptamers have

> been

> > identified which can bind to other nucleic acids (by means other

> than

> > conventional -Crick base pairing), proteins, small organic

> > compounds,

> > and even entire organisms. The binding agent (e.g. a nucleic acid

> probe

> > or

> > antibody) may be fixed to a solid support. The expression products

> may

> > then be

> > passed over the solid support, thereby bringing them into contact

> with

> > the

> > binding agent. Conveniently, the binding agents are immobilised at

> > defined,

> > spatially separated locations, to make them easy to manipulate

> during the

> > assay. The solid support may be a glass surface, e.g. a microscope

> slide,

> > beads, fibre-optics or microarray chip. In the case of beads, each

> > binding

> > agent may be fixed to an individual bead and they may then be

> contacted

> > with the

> > expression products in solution.

> >

> > [0118] The sample is generally contacted with the binding agent

> (s) under

> > appropriate conditions which allow the analyte in the sample to

> bind to

> > the

> > binding agent(s). The fractional occupancy of the binding sites of

> the

> > binding agent(s) can then be determined.

> >

> > [0119] Whatever the chosen assay system, there are numerous ways to

> > detect

> > interaction between the binding agent and the expression product

> > (analyte) to be

> > determined, either by directly or indirectly labelling " the

> analyte or

> > binding

> > agent, or by using a developing agent to arrive at an indication

> of the

> > presence

> > or amount of the analyte in the sample. A developing agent may be a

> > secondary

> > binding agent, capable of binding to a complex between analyte and

> > primary

> > binding agent. For example, if a primary antibody is used as a

> binding

> > agent,

> > the developing agent may be a secondary antibody capable of binding

> > either to

> > the primary antibody, or to a different epitope on the analyte to

> that

> > recognised by the primary antibody.

> >

> > [0120] Typically, the analyte, binding agent or developing agent is

> > directly or

> > indirectly labelled (e.g. with radioactive, fluorescent or enzyme

> labels,

> > such as horseradish peroxidase) so that they can be detected using

> > techniques

> > well known in the art. Directly labelled agents have a label

> associated

> > with or

> > coupled to the agent, Indirectly labelled agents may act on a

> further

> > species

> > to produce a detectable result. Thus, radioactive labels can be

> detected

> > using

> > a scintillation counter or other radiation counting device,

> fluorescent

> > labels

> > using a laser, confocal microscope, etc., and enzyme labels by the

> action

> > of

> > an enzyme label on a substrate, typically to produce a colour

> change. In

> > further embodiments, the developing agent or analyte is tagged to

> allow

> > its

> > detection, e.g. linked to a nucleotide sequence which can be

> amplified in

> > a

> > PCR reaction to detect the analyte. Other labels are known to those

> > skilled in

> > the art are discussed below.

> >

> > [0121] The developing agent (s) can be used in a competitive

> method in

> > which

> > the developing agent competes with the analyte for occupied

> binding sites

> > of the

> > binding agent, or non-competitive method, in which the labelled

> > developing agent

> > binds analyte bound by the binding agent or to occupied binding

> sites.

> > Both

> > methods provide an indication of the number of the binding sites

> occupied

> > by the

> > analyte, and hence the concentration of the analyte in the sample,

> e.g.

> > by

> > comparison with standards obtained using samples containing known

> > concentrations

> > of the analyte.

> >

> > [0122] In alternative embodiments, the analyte can be tagged before

> > applying it

> > to the support comprising the binding agent.

> >

> > [0123] There is an increasing tendency in the diagnostic field

> towards

> > miniaturisation of such assays, e.g. making use of binding agents

> (such

> > as

> > antibodies or nucleic acid sequences) immobilised in small,

> discrete

> > locations

> > (microspots) and/or as arrays on solid supports or on diagnostic

> chips.

> > These

> > approaches can be particularly valuable as they can provide great

> > sensitivity

> > (particularly through the use of fluorescent labelled reagents),

> require

> > only

> > very small amounts of biological sample from individuals being

> tested and

> > allow

> > a variety of separate assays can be carried out simultaneously.

> This

> > latter

> > advantage can be useful as it provides an assay employing a

> plurality of

> > analytes to be carried out using a single sample. Examples of

> techniques

> > enabling this miniaturised technology are provided in WO84/01031,

> > WO88/1058,

> > WO89/01157, WO93/8472, WO95/18376/ WO95/18377, WO95/24649 and EP 0

> 373

> > 203 A.

> >

> > [0124] Other methods which do not rely on labelling techniques may

> also

> > be used

> > to detect interaction between binding agent and reporter molecule,

> > including

> > physical methods such as surface plasmon resonance, agglutination,

> light

> > scattering or other means.

> >

> > [0125] Expressed nucleic acid (mRNA, pre-mRNA) can be isolated

> from the

> > cells

> > using standard molecular biological techniques. The expressed

> nucleic

> > acid

> > sequences corresponding to the gene or genes of Table 1 can then be

> > amplified

> > using nucleic acid primers specific for the expressed sequences in

> a PCR,

> > e.g.

> > real time PCR, multiplex PCR, etc. The skilled person will be able

> to

> > select

> > or design a suitable reaction type and protocol depending on, e.g.

> the

> > number

> > and particular combination of genes to be analysed. If the isolated

> > expressed

> > nucleic acid is mRNA, this can be converted into cDNA for the PCR

> > reaction using

> > standard methods.

> >

> > [0126] The primers may conveniently introduce a label into the

> amplified

> > nucleic

> > acid so that it may be identified. Ideally, the label is able to

> indicate

> > the

> > relative quantity or proportion of nucleic acid sequences present

> after

> > the

> > amplification event, reflecting the relative quantity or proportion

> > present in

> > the original test sample. For example, if the label is fluorescent

> or

> > radioactive, the intensity of the signal will indicate the

> relative

> > quantity/proportion or even the absolute quantity, of the expressed

> > sequences.

> > The relative quantities or proportions of the expression products

> of each

> > of the

> > genes of Table 1 may be used to establish a particular expression

> profile

> > for

> > the test sample.

> >

> > [0127] Other methods for detection of nucleic acid expression

> products

> > may also

> > be used, such as in situ hybridisation, Northern blot, etc.

> Likewise,

> > protein

> > expression products may be detected by any suitable technique.

> > Immunological

> > techniques are particularly preferred, in which antibodies

> specific for

> > the

> > particular polypeptide gene product (s), are used as binding

> agents,

> > although

> > other binding agents such as receptors or ligands capable of

> binding to

> > the

> > proteins of interest may be employed.

> >

> > [0128] In some embodiments, protein expression products from the

> sample

> > under

> > test are immobilised on a solid phase and contacted with a binding

> agent

> > specific for one or more of the proteins of Table 1 under

> appropriate

> > conditions

> > which allow binding between the protein and the binding agent. The

> amount

> > of

> > the binding agent found at the surface is then determined. For

> example,

> > the

> > binding agent may be directly labelled. Alternatively, the

> immobilised

> > antibody

> > may be contacted with a labelled developing agent capable of

> binding to

> > the

> > primary antibody. Examples of this type of assay include Western

> > blotting, and

> > certain ELISA (enzyme-linked immunosorbent assay) techniques.

> >

> > [0129] In other embodiments, a binding agent is immobilised on a

> solid

> > phase

> > and contacted with the sample under suitable conditions to allow

> binding

> > to take

> > place. The fractional occupancy of the binding sites of the

> binding agent

> > (s)

> > can then be determined either by directly or indirectly labelling

> the

> > analyte or

> > by using a developing agent or agents to arrive at an indication

> of the

> > presence

> > or amount of the analyte in the sample.

> >

> > [0130] An example of this type of assay is an antibody sandwich

> assay

> > (e.g. an

> > ELISA), which employs two antibodies each capable of binding to a

> > different

> > site on the biomarker protein. The first is immobilised on a solid

> phase

> > for use

> > as the binding agent. After contact with the analyte, the second

> antibody

> > is

> > used to detect complexes between the first antibody and analyte.

> > Whichever

> > method is chosen, it is important that the assay provides a read-

> out of

> > the

> > level of expression of the biomarker genes which allows results

> from

> > different

> > individuals to be compared reliably with one another. By way of

> example,

> > the

> > level of a particular expression product may be determined as a

> > proportion of

> > the total expression products found in the sample. Alternatively,

> the

> > level of

> > a particular expression product may be determined in relation to

> the

> > level of

> > expression of a control gene such as a housekeeping gene, or the

> like.

> > Alternatively, it may be convenient to determine the absolute

> amount of a

> > particular expression product, e.g. by comparison with known

> standards.

> > The

> > skilled person will be capable of designing a suitable protocol

> for any

> > given

> > assay method, and will also be aware of other suitable embodiments.

> >

> > [0131] Antibodies

> >

> > [0132] It has been shown that fragments of a whole antibody can

> perform

> > the

> > function of binding antigens. The term " antibody " is therefore used

> > herein to

> > encompass any molecule comprising the binding fragment of an

> antibody,

> > and the

> > term binding agent and binding site should be construed

> accordingly.

> > Examples

> > of binding fragments are (i) the Fab fragment consisting of VL,

> VH, CL

> > and CHl

> > domains; (ii) the Fd fragment consisting of the VH and CHl

> domains; (iii)

> > the

> > Fv fragment consisting of the VL and VH domains of a single

> antibody;

> > (iv) the

> > dAb fragment (Ward, E.S. et al., Nature 341, 544-546 (1989)) which

> > consists of a VH domain; (v) isolated CDR regions; (vi) F (ab ') 2

> > fragments,

> > a bivalent fragment comprising two linked Fab fragments (vii)

> single

> > chain Fv

> > molecules (scFv), wherein a VH domain and a VL domain are linked

> by a

> > peptide

> > linker which allows the two domains to associate to form an antigen

> > binding site

> > (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA,

> 85,

> > 5879-5883,

> > 1988). In preferred embodiments the binding agent comprises a

> single

> > antigen

> > binding site specific for the analyte, i.e. a monovalent antibody

> or

> > antibody

> > fragment.

> >

> > [0133] Pha rmaceutical compositions Pharmaceutical compositions as

> > described in

> > this specification typically comprise, in addition to one or more

> > suitable

> > active agents, a pharmaceutically acceptable excipient, carrier,

> buffer,

> > stabiliser or other materials well known to those skilled in the

> art.

> > Such

> > materials should be non-toxic and should not interfere with the

> efficacy

> > of the

> > active ingredient. The precise nature of the carrier or other

> material

> > may

> > depend on the route of administration, e.g. oral, intravenous,

> cutaneous

> > or

> > subcutaneous, nasal, intramuscular, intraperitoneal routes.

> >

> > [0134] Pharmaceutical compositions for oral administration may be

> in

> > tablet,

> > capsule, powder or liquid form. A tablet may include a solid

> carrier such

> > as

> > gelatin or an adjuvant. Liquid pharmaceutical compositions

> generally

> > include a

> > liquid carrier such as water, petroleum, animal or vegetable oils,

> > mineral oil

> > or synthetic oil.

> >

> > [0135] Physiological saline solution, dextrose or other saccharide

> > solution or

> > glycols such as ethylene glycol, propylene glycol or polyethylene

> glycol

> > may be

> > included.

> >

> > [0136] For intravenous, cutaneous or subcutaneous injection, or inj

> > ection at

> > the site of affliction, the active ingredient will be in the form

> of a

> > parenterally acceptable aqueous solution which is pyrogen-free and

> has

> > suitable

> > pH, isotonicity and stability. Those of relevant skill in the art

> are

> > well able

> > to prepare suitable solutions using, for example, isotonic

> vehicles such

> > as

> > Sodium Chloride Inj ection, Ringer ' s Inj ection, Lactated

> Ringer ' s

> > Inj

> > ection. Preservatives, stabilisers, buffers, antioxidants and/or

> other

> > additives may be included, as required.

> >

> > [0137] Whether it is a polypeptide, antibody, peptide, nucleic acid

> > molecule,

> > small molecule or other pharmaceutically useful compound that is

> to be

> > given to

> > an individual, administration is preferably in a " prophylactically

> > effective

> > amount " or a " therapeutically effective amount " (as the case may

> be,

> > although

> > prophylaxis may be considered therapy), this being sufficient to

> show

> > benefit

> > to the individual. The actual amount administered, and rate and

> > tir[alpha]e-

> > course of administration, will depend on the nature and severity

> of what

> > is

> > being treated. Prescription of treatment, e.g. decisions on dosage

> etc,

> > is

> > within the responsibility of general practitioners and other

> medical

> > doctors,

> > and typically takes account of the disorder to be treated, the

> condition

> > of the

> > individual patient, the site of delivery, the method of

> administration

> > and other

> > factors known to practitioners. Examples of the techniques and

> protocols

> > mentioned above can be found in Remington' s Pharmaceutical

> Sciences,

> > 20th

> > Edition, 2000, pub. Lippincott, & Wilkins.

> >

> > [0138] Alternatively, targeting therapies may be used to deliver

> the

> > active

> > agent more specifically to certain types of cell, by the use of

> targeting

> > systems such as antibody or cell specific ligands. Targeting may be

> > desirable

> > for a variety of reasons; for example if the agent is unacceptably

> toxic,

> > or if

> > it would otherwise require too high a dosage, or if it would not

> > otherwise be

> > able to enter the target cells.

> >

> > [0139] A composition may be administered alone or in combination

> with

> > other

> > treatments, either simultaneously or sequentially dependent upon

> the

> > condition

> > to be treated.

> >

> > [0140] Examples A group at the University of UIm, Germany, has

> recently

> > suggested that a pentapeptide (QYNAD) with Na<+> channel-blocking

> > function could

> > be a biological marker of certain inflammatory and immunological

> > disorders of

> > the nervous system<16>.

> >

> > [0141] The inventors asked whether or not the pentapeptide

> identified by

> > the

> > German group<16> might play a role in CFS. Samples of serum were

> sent to

> > the

> > University of UIm for analysis. The 15 samples included 5 normal

> > controls, 5

> > patients with CFS and 5 disease controls including two patients

> with MS.

> > Samples were numbered 1-15 and the German group were not informed

> what

> > the

> > samples were, or which samples were which, until the experiment was

> > concluded.

> >

> > [0142] When the code was broken, the results showed that the

> disease

> > control

> > group had levels of the pentapeptide which were 2.3X those of the

> normal

> > controls (similar to the published data) and the CFS samples had

> levels

> > which

> > were 3X higher than the healthy controls.

> >

> > [0143] Thus, there are measurably higher amounts of the

> pentapeptide in

> > patients with CFS compared with healthy controls. Although the

> > pentapeptide may

> > not be specific to CFS (as high levels are also found in other

> > disorders), an

> > assay for the peptide could be used as part of the differential

> diagnosis

> > of CFS.

> >

> > [0144] The German group was unable to identify an endogenous gene

> which

> > encodes

> > the pentapeptide. The inventors carried out NCBI BLAST and

> > EMBL-Heidelberg

> > Bioccelerator amino acid alignments for the pentapeptide QYNAD. A

> total

> > of one

> > hundred alignment hits were found. Of these, only nine showed 100%

> > similarity

> > over the five amino acids - five of those were human. The amino

> acid

> > searches

> > were followed by NCBI BLAST searches using the GenBank Accession

> and gi

> > numbers

> > for each of the five human amino acid to determine their origins,

> > references

> > and nucleotide sequences. A number of cloned nucleotide sequences

> were

> > found

> > and when these were run through the nucleotide databases, only one

> clone

> > showed

> > full-length homology to any human gene. This gene was a human ion-

> channel

> > gene

> > - the vacuolar proton pump H<+>-ATPase (v-ATPase). Remarkably,

> when the

> > human

> > gene amino acid sequence was compared with the original QYNAD

> > pentapeptide it

> > was discovered that the relevant part of the human ion channel

> encodes

> > the

> > sequence QYMAD.

> >

> > [0145] The inventors next asked whether the v-ATPase represents a

> > candidate gene

> > for a diagnostic test for CFS. RT-PCR using primers specific for

> the

> > v-ATPase

> > was performed on cDNA prepared from mRNA from PBMCs from CFS

> patients and

> > healthy controls. As shown in Figure 1, the patient samples have a

> > significantly higher level of v-ATPase mRNA than the healthy

> controls.

> > Thus the

> > v-ATPase gene appears to represent a genuine biomarker for CFS/ME.

> >

> > [0146] The vATPase is known to be involved in regulation of a

> number of

> > metabolic functions which are deranged in CFS/ME. vATPase

> upregulation

> > could

> > therefore provide an explanation for a number of the symptoms

> observed.

> > For

> > example, increased vATPase activity could explain the intracellular

> > acidosis in

> > exercising muscles, chest pain (syndrome X), altered

> neurotransmitter

> > (dopamine) function and abnormal regulation of hypothalamic

> hormones. In

> > addition, it could explain the increased energy expenditure and

> fatigue

> > associated with the condition. Taken together, this suggests that

> the

> > vATPase is

> > not only a marker for the condition, but is a realistic target for

> > intervention

> > therapy.

> >

> > [0147] Other biomarkers for CFS/ME

> >

> > [0148] The inventors went on to examine whether the increase in

> vATPase

> > expression was confirmed by microarray analysis. Such analysis

> provides

> > the

> > opportunity to examine the differential expression of mRNA from a

> very

> > large

> > number of genes. Surprisingly, the results of the analysis not only

> > confirmed

> > their earlier findings regarding the vATPase gene, but also

> identified

> > differences in the level of expression of key genes in the PBMC of

> > patients with

> > CFS/ME and control subj ects, giving an insight into the

> biochemical

> > pathways

> > which are involved in this disorder.

> >

> > [0149] Microarray results have been verified by western blot

> analysis and

> > RT-PCR

> > assay. A number of genes, in addition to v-ATPase, were

> significantly

> > up/downregulated and identified as suitable biomarkers for the

> disorder.

> >

> > [0150] Advances in genome sequencing and automated chip

> manufacture have

> > made

> > DNA chip or microarray technology readily available<25>. This

> technology

> > allows

> > simultaneous differential expression profiling from a very large

> number

> > of genes

> > in tissue samples of CFS/ME patients and controls. A recent report

> from

> > Vernon

> > et al (2002), described a CFS biomarker search in PBMC using a DNA

> chip

> > array

> > assay which included 1, 764 genes. In the study reported here, RNA

> > isolated

> > from PBMC, was assayed using Affymetrix genome-wide chips (HG-U133

> > arrays)

> > which included 30,000 gene sequences.

> >

> > [0151] Using DNA microarray analysis of whole human genome, gene

> > transcriptional signatures were compared in the PBMC of eight male

> > patients with

> > CFS and seven age-matched male healthy controls. An additional

> cohort of

> > fourteen patients with CFS and age and sex matched controls was

> recruited

> > for

> > RT-PCR and western blot assays in order to verify the microarray

> data.

> > Analysis

> > of the microarray data was performed as described previously

> (Breitling

> > R.

> > Armengaud P. Amtmann A. Herzyk P. Rank products: A simple, yet

> powerful,

> > new

> > method to detect differentially regulated genes in replicated

> microarray

> > experiments. FEBS Letters 2004; 573 (1-3): 83-92; Breitling R.

> Amtmann A.

> > Herzyk P. Iterative Group Analysis (iGA): A simple tool to enhance

> > sensitivity

> > and facilitate interpretation of microarray experiments. BMC

> > Bioinformatics

> > 2004; 5: (pp 8p)).

> >

> > [0152] Genes which are significantly upregulated in CFS/ME patients

> > compared to

> > healthy controls are detailed in Table 6, ranked according to

> their RP

> > values.

> > It is considered that any of these genes may be used as biomarkers

> for

> > CFS/ME.

> > Particularly preferred marker genes are detailed in Table 1.

> >

> > [0153] Further genes, including prostaglandin D2 synthase and T-

> cell

> > receptors

> > alpha, beta, gamma and delta are found to be downregulated in CFS

> > patients

> > compared to normal controls. Prostaglandin D2 synthase (NCBI

> accession

> > no. BC

> > 005939, UniGene Hs. 446429) is considered to be a good candidate

> for a

> > CFS/ME

> > biomarker, because it is known to be involved in sleep regulation;

> > patients with

> > CFS frequently suffer from sleep reversal and fatigue associated

> with

> > lack of

> > sleep. However, data for other downregulated genes is not shown

> here. In

> > general, genes which are upregulated in the disease state are

> considered

> > to be

> > better biomarkers for diagnostic tests etc. than genes which are

> > downregulated

> > because the potential for false-positive tests is significantly

> higher

> > when

> > using genes which are underexpressed in the disease state.

> >

> > [0154] Iterative group analysis of the differentially expressed

> genes

> > indicate

> > that in CFS, there is a shift of immune response with preferential

> > antigen

> > presentation to MHC class Il receptors and downregulation of T-

> cell

> > receptor-[alpha], increased cell membrane prostaglandin-

> endoperoxide

> > synthase

> > activity with downstream changes in oxygen transport and also

> activation

> > of the

> > guanyl cyclase and caspase pathways of cellular apoptosis. Another

> set of

> > genes

> > was identified which are involved in the immediate response to

> infection,

> > particularly by intracellular parasites. The particular genes

> involved in

> > each

> > of these pathways are identified in Tables 2 to 5. In each of

> these key

> > pathways,

> > the hub genes were higher ranked in the analysis compared to the

> network

> > genes.

> >

> > [0155] Functional changes produced by altered gene regulation may

> explain

> > the

> > mechanism of fatigue and offer a rational basis for targeted

> > pharmacotherapy in

> > CFS.

> >

> > [0156] Table 1 - Biomarker Genes for CFS/ME

> >

> > [0157]

> > <EMI ID=36.1>

> >

> >

> > [0158]

> > <EMI ID=37.1>

> >

> > [0159] Table 2: Genes involved in defence against infection by

> > intracellular

> > pathogens

> >

> > [0160]

> > <EMI ID=37.2>

> >

> > [0161]

> > <EMI ID=38.1>

> >

> > [0162] Table 3: Genes involved in oxidative stress response

> >

> > [0163]

> > <EMI ID=38.2>

> >

> > [0164] Table 4: Genes involved in apoptosis

> >

> > [0165]

> > <EMI ID=38.3>

> >

> > [0166] Table 5: Genes involved in antigen presentation and immunity

> >

> > [0167]

> > <EMI ID=38.4>

> >

> > [0168]

> > <EMI ID=39.1>

> >

> > [0169] NCBI accession numbers refer to the UniGene database, build

> no.

> > 177,

> > released 22 December 2004.

> >

> > [0170] It is clear from this data that significant differences in

> the

> > expression

> > of a number of genes can be seen in PBMC samples from patients

> with CFS

> > and

> > healthy controls. We have verified that the DNA microarray assay

> is valid

> > by

> > confirming the results by RT-PCR and western blot analyses. This

> is the

> > first

> > time that a reproducible biochemical lesion has been seen in

> patients

> > with CFS.

> > We propose that bioassays of the significantly over-expressed

> genes could

> > be

> > used as diagnostic biomarkers for CFS to aid in the differential

> > diagnosis of

> > the condition.

> >

> > [0171] In order to confirm the relevance of the genes identified

> in the

> > microarray experiments, Western blot and RT-PCR assays have been

> > performed to

> > analyse the expression of selected genes in samples from patients

> and

> > controls

> > different to those studied in the microarray analysis. The results

> verify

> > that

> > these genes may be used as potential biomarkers to support the

> clinical

> > diagnosis of CFS and identify suitable candidates for treatment

> trials.

> >

> > [0172] Figures 2 to 5 show upregulation of mRNA and/or proteins

> from

> > various of

> > these genes in PBMCs from patients suffering from CFS/ME, as

> compared to

> > age-

> > and sex-matched healthy controls. Figure 2 shows RT-PCR analysis of

> > Defensin

> > alpha 1, Figures 3 and 4 show Western blot analysis of Defensin

> alpha 1

> > and

> > Thrombospondin 1 respectively. In all cases, there is significant

> > upregulation

> > of the protein or mRNA in the disease group compared to the control

> > group.

> > Figure 6 shows a duplicate of the SDS-PAGE gel used for the

> Western blot,

> > stained with Coomassie blue. This confirms that the observed

> differences

> > in

> > protein levels between the disease and control groups are not due

> to

> > unequal

> > loading of total protein on the gels.

> >

> > [0173] Discussion

> >

> > [0174] Previous reports have hypothesised that CFS is a form of

> > channelopathy -

> > a disorder of membrane ion channels<9>' <11>' <13>. There are

> several

> > reports in

> > the literature which we believe strengthen the hypothesis that the

> > vacuolar H+

> > ATPase plays a pathogenic role in CFS.

> >

> > [0175] Local anaesthetics, which are known to act on ion channels,

> have

> > an

> > adverse effect on patients with CFS/ME. It has been demonstrated

> also,

> > that in

> > some patients with CFS/ME, there are morphological changes to the

> red

> > blood

> > cells<19>. Remarkably, a study by Nishiguchi et al<20>, has

> demonstrated

> > that

> > the local anaesthetic lidocaine can induce reversible

> morphological

> > transformation of human red blood cells and that this change is

> mediated

> > by the

> > activation of vacuolar H+ ATPase. In addition, Li et al<21a> have

> shown

> > that

> > the gene is involved in iron binding in red blood cells.

> >

> > [0176] The ion channel gene is a member of the vacuolar H+ ATPase

> proton

> > transporting gene family<21>'<22>'<23>. This family of genes is

> directly

> > involved with the phosphocreatine-dependent glutamate uptake by

> synaptic

> > vesicles<24>. The gene is responsible for vesicle

> docking/exocytosis

> > during

> > neurotransmiter release<25> and is a major constituent of synaptic

> > vesicles

> > associated with intracellular membrane structures<26>. We have

> > demonstrated,

> > using <1>H MRS that there is a perturbation of the choline/creatine

> > balance in

> > the CNS (Condon et al<17>, Chaudhuri et al<42>). This finding has

> been

> > corroborated by Puri et al<18>. As stated above, this type of gene

> is

> > directly

> > involved in the creatine pathways. We have previously demonstrated

> that

> > patients with CFS have low body- potassium levels<9>. et

> al<27>

> > have

> > shown a relationship between potassium depletion and up-regulation

> of

> > H+-ATPase.

> >

> > [0177] As stated above, viruses have often been associated with

> CFS.

> > Virus

> > entry into cells may be mediated by H+ATPase<28>'<29>'<30>. In

> addition

> > to viral

> > infection affecting neurotransmitter function<7>, there is a large

> body

> > of

> > evidence to show that the vacuolar H+-ATPase is also

> > invoked<31>'<32>'<33,34>'

> > <35>'<36>'<37>'<38, 39.>

> >

> > [0178] It is clear from the above data that significant

> differences in

> > the

> > expression of a number of genes can be seen in PBMC samples from

> patients

> > with

> > CFS and healthy controls. This is the first time that a

> reproducible

> > biochemical lesion has been seen in patients with CFS We propose

> that

> > bioassays

> > of the significantly over-expressed genes (below) could be used as

> > diagnostic

> > biomarkers for CFS to aid in the differential diagnosis of the

> condition.

> >

> > [0179] Materials and Methods

> >

> > [0180] Study Subjects

> >

> > [0181] Patients with CFS were diagnosed with reference to the 1994

> Fukuda

> > definition. All seven patients were male, aged between 18 and 54

> years

> > (mean

> > 36), and were not on medication. Healthy control subj ects were

> male,

> > aged

> > between 22 and 58 years (mean 34). In addition to the 8 patients

> and

> > seven

> > control subjects used for the DNA chip assays, an additional

> fourteen

> > patients

> > and controls were used to confirm the chip assay results by RT-PCR

> and

> > western

> > blot assays. Informed consent and ethical approval were obtained.

> >

> > [0182] Isolation of peripheral blood mononuclear cells (PBMC) for

> protein

> > purification

> >

> > [0183] Venous blood samples were drawn from patients who fulfilled

> the

> >

> > [0184] Holmes and Fukuda criteria for ME/CFS, and from healthy

> > individuals. The

> > procedure for isolating PBMC was started immediately and finished

> within

> > 2h of

> > sampling. EDTA treated whole blood was diluted 1: 1 with phosphate

> > buffered

> > saline. Two volumes of blood were overlaid on one volume of

> Histopaque

> > -1077

> > (Sigma Diagnostics) and centrifuged at 20[deg.]C AT 50Og for 30

> min. The

> > PBMC

> > interface was removed and washed twice with phosphate buffered

> saline and

> > centrifuged. The pellets were resuspended in phosphate buffered

> saline,

> > an

> > aliquot removed and counted in red blood cell lysis buffer (155Mm

> NH4CL,

> > 10Mm

> > NaHCO3, pH7.4, 0.1mM EDTA). The PBMC were centrifuged once more

> > (20[deg.]C,

> > 500g, 10min). The PBMC were then aliquoted into micortubes

> equivalent to

> > 5x105

> > cells per tube, centrifuged and stored as dry pellets at -80[deg.]

> C.

> >

> > [0185] Western Blot Analysis

> >

> > [0186] PBMC pellets were resuspended in sample reducing buffer (ImI

> > Glycerol,

> > 0.5ml [beta]-mercaptoethanol, 3ml 10% SDS, 1.25ml IM Tris-HCL Ph

> 6.7),

> > boiled

> > for 5 min. The lysates were loaded onto a 10% PAGE gel, each track

> > equivalent

> > to 2x 10 5 PBMC. The PAGE gel was assessed for equal protein load

> by

> > coomassie

> > stain. The gel then electrophoretically transferred onto

> nitrocellulose

> > PVDF

> > membrane (Biorad) for 2 hours. The blots were blocked for non

> specific

> > binding

> > with 10% normal goat serum for 30 min, probed with a mouse Mab to

> human

> > defensinl-3, (Hycult, Netherlands), mouse Mab to human

> thrombospondin

> > (Sigma-Aldrich Inc) and a mouse Mab to Chondroitin Sulphate

> Proteoglycan

> > (USBiological MA USA), at dilutions of 1/100, 1/1000, 1/1000

> respectively

> > in

> > TBS 0.05% Tween 20 for 2 hours at RT. The protein was detected

> after

> > subsequent

> > incubation with alkaline phosphatase conjugate Goat anti Mouse IgG

> > (1/1000

> > final dilution) ( Immunoresearch Laboratories PA USA). The

> > reactions

> > were detected using SIGMA FAST BCIP/NBT).

> >

> > [0187] Development of the PCR assay for the CFS gene.

> >

> > [0188] Oligonucleotide primers which span a specific epitope

> within the

> > CFS gene

> > were chosen and tested by RT-PCR. RNA from blood samples from

> patients

> > with CFS

> > and appropriate controls were RT-PCR amplified and PCR amplicons

> > quantitated by

> > gel documentation system software.

> >

> > [0189] RNA Isola tion for RT-PCR

> >

> > [0190] Total RNA was isolated from peripheral blood mononuclear

> cells

> > (PBMC)

> > using the Promega RNAgents Total RNA Isolation System.

> >

> > [0191] Venous blood was collected in standard EDTA blood tubes and

> RNA

> > purified

> > using the method of Chomczynski and Sacchi. White blood cell

> pellets were

> > homogenised by hand in an appropriate volume of denaturing solution

> > (guanidinium

> > thiocyanate, 4M; sodium citrate, 25mM; N-laurolyl sarcosine, 0.5%;

> > 2-mercaptoethanol, 0.1M, in distilled water adjusted to pH 7.0). To

> > homogenate

> > (ImI), sodium acetate (100[mu]l, 2M, pH 4.0), citrate-buffered

> (0.1M, pH

> > 4.3) phenol

> >

> > [0192] { lml) and chloroform: isoamyl alcohol (49: 1, 200[mu]l)

> were

> > sequentially added. The resulting mixture was treated in a vortex

> mixer

> > (Fisons

> > Scientific Equipment, Whirlimixer) for 10 seconds then

> incubated on

> > ice for

> > 15 minutes. Samples were then centrifuged (12, 000g, 20 minutes,

> > 4[deg.]C) and

> > the upper aqueous layer pippetted into to a fresh tube. After

> addition of

> > ice

> > cold isopropanol (lml), RNA precipitated from the mixture during a

> thirty

> > minute period on dry ice. The mixtures were then centrifuged (12,

> 000g,

> > 20

> > minutes, 4[deg.] C) and the supernatant discarded. The pellet was

> > dissolved in

> > denaturing solution (300[mu]l) and transferred to a

> microcentrifuge tube (

> > 1.5ml, Axygen). Ethanol (absolute, 600[mu]l) was added to each

> > microcentrifuge

> > tube, samples were incubated on dry ice for 30 minutes, and then

> > centrifuged (

> > 1160Og, 20 minutes, 4[deg.]C). The resulting pellet were then

> washed

> > twice in

> > ethanol (70% aqueous solution) then lyophylised (using a Hetosicc

> > freeze-drier

> > and a JAVAC high vacuum pump DD-75). The freeze-dried RNA was

> > re-suspended in

> > sterile distilled water (60[mu]l) and stored at -70[deg.]C until

> required.

> >

> > [0193] Quantifica tion and examination of RNA

> >

> > [0194] RNA solution (prepared as described above, 2[mu]l) was

> added to

> > distilled

> > water (98[mu]l) to produce a 1: 50 dilution. The optical density

> of the

> > sample was read at 260nn[alpha] and 280nm using a 50[mu]l

> ultraviolet

> > cuvet.

> > The absorbance ratio 260/280nm measured at these wavelengths

> indicates

> > the

> > purity of the RNA. The absolute concentration of RNA was estimated

> using

> > the

> > following equation:

> >

> > [0195] Optical Density260nm x 40 x dilution = RNA concentration

> ([mu]g/ml)

> >

> > [0196] To examine the quality, 5[mu]l of RNA was added to 5[mu]l

> of the

> > gel

> > marker Orange G (1% Orange G dye in 50% glycerol, 50% 2x TBE).

> This was

> > heated

> > to 70[deg.]C for 3 minutes, then electrophoresed through a

> horizontal 1%

> > agarose gel in 1 x TBE (10x TBE = 0.089M tris (hydroxymethyl) -

> > methylamine,

> > 0.089M boric acid, 0.025M disodium EDTA, pH 8.3). The gel was then

> > stained for

> > 30 minutes in ethidium bromide (0.5 mg/ml) in TBE, de-stained in

> water

> > and

> > visualised under medium wave (320nm) ultraviolet light.

> >

> > [0197] cDNA synthesis

> >

> > [0198] Two micrograms of RNA were added to 1[mu]l oligo (dT) 12-18

> (

> > 500[mu]g/ml, Roche) and the volume made up to 11[mu]l with sterile

> > distilled

> > water.

> >

> > [0199] This was incubated (70[deg.]C, 10 minutes) to allow the

> oligo (dT)

> > to

> > bind to the poly-A tail of the RNA, and then chilled on ice. To the

> > reaction

> > mixture, 4[mu]l of 5x First Strand Buffer, 2[mu]l of 0. IM DTT, 1

> [mu] l

> > of 10mM

> > dNTP mix (10mM each dATP, dGTP, dCTP, dTTP; Amersham Pharmacia

> Biotech),

> > 1[mu]l of sterile distilled water and 1[mu]l of

> >

> > [0200] Superscript II (Gibco-BRL® Life Technologies) were added.

> This

> > was

> > incubated first at 50[deg.]C for Ihr, then 70[deg.]C for 15

> minutes to

> > inactivate the reaction. A negative cDNA control (2[mu]l of

> distilled

> > water)

> > was included in each cDNA synthesis to confirm that the reaction

> mix was

> > not

> > contaminated.

> >

> > [0201] Polymerase Chain Reaction To 2[mu]l of cDNA, 10[mu]l of 10x

> > magnesium-free buffer, 10[mu]l of 2.5mM dNTPs (2.5mM each dATP,

> dGTP,

> > dCTP,

> > dTTP), 6[mu]l MgCl2 (25mM) and 1[mu]l each of the appropriate 5'

> and 3 '

> > primers (0.5[mu]g/[mu]l) were added. The volume was made up to 99.8

> [mu]l

> > using

> > sterile distilled water and 0.2[mu]l of Taq DNA polymerase

> (Promega) was

> > added.

> > The PCR reaction (35 cycles) was carried out on a Techne Genius

> > thermocycler.

> >

> > [0202] Primers used to amplify vATPase were 5 ' -etc gtg ace tgt

> tac tgc

> > tg - 3

> > ' and 5 ' -aag taa cca agt cca etc ca-3 '. Primers for Defensin 1

> were 5

> > ' -caa

> > gag ctg atg agg ttg ct-3 ' and 5 ' -gaa ggt aca gga gta ata gc-3 '.

> >

> > [0203] Thirty microlitres of PCR product was added to 5[mu]l of

> orange G

> > and

> > electrophoresed through a horizontal 2-3% agarose gel in 1 x TBE

> at 100

> > volts,

> > until the dye front had migrated a minimum of 10cm. On each gel, 3

> [mu]l

> > of a 123

> > base pair DNA ladder in 5[mu]l of Orange G was included as a size

> marker

> > for

> > comparison with PCR product bands. The gel was then stained for 30

> > minutes in

> > ethidium bromide (0.5 mg/ml) in TBE, destained in water and

> visualised

> > under

> > medium wave (320nm) ultraviolet light.

> >

> > [0204] Gene expression was analysed by measuring the band density

> for

> > each

> > amplicon. Band densities were measured using the Herolab EASY Plus

> > computer

> > automated image analysis system. True comparisons in gene

> expression

> > between

> > samples were enabled by comparing results of densitometry for the

> > experimental

> > genes against the housekeeping gene (abl - tyrosine kinase) band

> > densities.

> > References

> >

> > [0205] 1. Chaudhuri A, Behan PO. Neurological dysfunction in

> chronic

> > fatigue

> > syndrome. J Chr Fatigue Synd 2000; 6 (3/4): 51-68

> >

> > [0206] 2. Fukuda K, Strauss SE, Hickie I, et al. The chronic

> fatigue

> > syndrome:

> > a comprehensive approach to its definition and study. Ann Intern

> Med

> > 1994;

> > 121: 953-59

> >

> > [0207] 3. The National Task Force Report on CFS/ME (30 September

> 1998).

> >

> > [0208] 4. Chaudhuri A, Behan WMH, Behan PO. Chronic fatigue

> syndrome.

> > Proc R

> > Coll Physicians Edinb 1998: 28: 150-63 5. Chaudhuri A, Gow J,

> Behan PO.

> > Systemic viral infections and chronic fatigue syndrome: current

> concept

> > and

> > recent advances. In: Abramsky O, D (eds). Brain disease:

> > Therapeutic

> > strategies and repair. London: Dunitz 2001 (in press)

> >

> > [0209] 6. de Ia Torre JC, Borrow P, Oldstone MBA. Viral

> persistence and

> > disease:

> > cytopathology in the absence of cytolysis. Br Med Bull

> >

> > [0210] 1991; 47: 838-51

> >

> > [0211] 7. Gies U, Bilzer T, Stitz L, Staiger JF. Disturbance of the

> > cortical

> > cholinergic innervation in Borna disease prior to encephalitis.

> Brain Pa

> > thology 1998; 8: 39-48 8. Kress M, Fickenscher H. Infection by

> human

> > varicella-zoster virus confers norepinephrine sensitivity to

> sensory

> > neurons

> > from rat dorsal root ganglia. FASEB Journal 2001; 15: 1037-43

> >

> > [0212] 9. Chaudhuri A, WS, Behan PO. Arguments for a role of

> > abnormal

> > ionophore function in chronic fatigue syndrome. In: Yehuda S,

> Moltofsky

> > DI

> > (eds). Chronic fatigue syndrome. New York, Plenum Press 1997; 119-

> 30

> >

> > [0213] 10. Goncealves PP, Meireles SM, Neves P, Vale MGP. Ca<2+>

> > sensitivity of

> > synaptic vesicle dopamine, gamma-aminobutyric acid and glutamate

> > transport

> > system. Neurochemical research 2001; 26: 75-81

> >

> > [0214] 11. Gow JW, McGarry F, Behan WMH, Simpson K, Behan PO.

> Molecular

> > analysis

> > of cell membrane ion channel function in chronic fatigue syndrome

> > (Abstract).

> > International Meeting on Chronic Fatigue Syndrome, Dublin 1994. 12.

> > Ackerman MJ,

> > Clapham DE. Ion channels-basic science and clinical diseases. N

> Engl J

> > Med

> > 1997; 336: 1575-86

> >

> > [0215] 13. Chaudhuri A, WS, Pearn J, Behan PO. Symptoms of

> chronic

> > fatigue syndrome are due to abnormal ion channel function. Medical

> > Hypotheses

> > 2000; 54: 59-63.

> >

> > [0216] 14. Pearn J. Chronic ciguatera: one organic cause of the

> chronic

> > fatigue

> > syndrome. J Chr Fa tigue Synd 1996; 2: 29-34

> >

> > [0217] 15. Bidard JN, Viverberg HPM, Frelin C. Ciguatoxin is a

> novel type

> > of

> > Na<+> channel toxin. J Biol Chem 1984; 259: 8353-57 16. Brinkmeier

> H,

> > Aulkemeyer P, Wollinsky KH, Rudel R. An endogenous pentapeptide

> acting as

> > a

> > sodium channel blocker in inflammatory autoimmune disorders of the

> > central

> > nervous system. Nature Med 2000; 6: 808-11.

> >

> > [0218] 17. Condon BR, Chaudhuri A, Hadley DN, Brennan D, (2002),

> > " Increased

> > levels of choline containing compounds found in the basal ganglia

> of

> > patients

> > with chronic fatigue syndrome " in Proc Intl. Soc. Mag. Reson. Med.

> 10.

> >

> > [0219] 18. Puri BK, et al, (2002), " Relative increase in choline

> in the

> > occipital cortex in chronic fatigue syndrome " Acta Psychiatr Scand

> 106

> > (3):

> > 224-226.

> >

> > [0220] 19. Simpson LO. (1992) " The role of nondiscocytic

> erythrocytes in

> > the

> > pathogenesis of myalgic encephalomyelitis/chronic fatigue

> syndrome " In

> > Hyde BM,

> > Goldstein, Levine P (eds). The clinical and scientific basis of

> myalgic

> > encephalomyelitis/chronic fatigue syndrome. The Nightingale

> Research

> > Foundation, Canada. 597-605.

> >

> > [0221] 20. Nishiguchi E, Hamada N, Shindo J. (1995), " Lidocaine

> action

> > and

> > conformational changes in cytoskeletal protein network in human

> red blood

> > cells "

> > Eur J Pharmacol, 286 (1): 1-8. 21. Forgac, M. Structure and

> properties of

> > the vacuolar (H<+>) - ATPases-. J. Biol. Chem. 274 (19), 12951-

> 12954

> > (1999).

> > 21a. Li, C-Y., Watkins, J.A., Hamazaki, S., Altazan, J. D. &

> Glass, J.

> > Iron binding, a new function for the reticulocyte endosome H+-

> ATPase.

> > Biochemistry. 34 (15), 5130-5136 (1995). 22. s, T. H. &

> Forgac,

> > M. Structure, function and regulation of the vacuolar (H<+>) -

> ATPase.

> > Annu.

> > Rev. Cell Dev. Biol 13, 779-808

> >

> > [0222] (1997).

> >

> > [0223] 23. , N. & Harvey, W. R. Vacuolar and plasma membrane

> > proton-

> > adenosinetriphosphatases. Physiol. Rev. 79 (2), 361-385 (1999).

> >

> > [0224] 24. Xu CJ, Klunk WE, Kanfer JN, et al, (1996),

> Phosphocreatine-

> > dependent glutar[alpha]ate uptake by synaptic vesicles " J

> Biological

> > Chemistry,

> > 271 (23): 13435-13440.

> >

> > [0225] 25. Siebert A, Lottspeich F, N, Betz H (1994).

> Purification

> > of

> > the synaptic vesicle-binding protein physophilin.

> >

> > [0226] Identification as 39-kDa subunit of the vacuolar H+-

> ATPase. J.

> > Biological Chemistry. 269 (45): 28329-28334.

> >

> > [0227] 26. Brochier G, Morel N (1993). The same 15kDA proteolipid

> subunit

> > is a

> > constituent of two different proteins in Torpedo, the acetycholine

> > releasing

> > protein and the vacuolar H+ ATPase. Neurochem. International. 23

> (6):

> > 525-539.

> >

> > [0228] 27. , M., Capasso, G., Agulian, S., Giebisch, G. &

> Unwin, R.

> > The relationship between distal tubular proton secretion and

> dietary

> > potassium

> > depletion: evidence for up-regulation of H+- ATPase. Nephrol.

> Dial.

> > Transplant. 14 (6), 1435-1440 (1999).

> >

> > [0229] 28. Hansen, J., Qing, K. & Srivastava, A. Adeno-associated

> virus

> > type 2-medicated gene transfer: Altered endocytic processing

> enhances

> > transduction efficiency in murine fibroblasts. J. Virol. 75 (9),

> 4080-4090

> > (2001). 29. Suzuki, T. Yamayam M, Sekizawa, K., Hosada, M.,

> Yamada, N.,

> > Ishizuka, S., Nakayama, K., Yanai, M., Numazaki, Y., Sasaki, H.

> > Bafilomycin A. K inhibits rhinovirus infection in human airway

> epithelium:

> > Effects on endosome and ICAM-I. Am. J. Physiol. Lung Cell. MoI.

> Physiol.

> > 280 (6 24-6), L1115-L1127 (2001). 30. Guinea, R. & Carrasco, L.

> > Requirement for vacuolar proton- ATPase activity during entry

> influenza

> > virus into cells. J. ViroJ. 69 (4), 2306-2312 (1995).

> >

> > [0230] 31. Moriyama, Y. & Futai, M. Presence of 5-

> hydroxytryptamine

> > (serotonin) transport coupled with vacuolar-type H+ATPase in

> > neuroscretory

> > granules from bovine posterior pituitary. J. Biol. Chem. 265 (16),

> > 9165-9169

> > (1990).

> >

> > [0231] 32. Wolosker, H., de Souza, D. O. & de Meis, L. Regulation

> of

> > glutamate transport into synaptic vesicles by chloride and proton

> > gradient. Am.

> > Soc. Biochem. MoI. Bio. 271 (20), 11726-11731 (1996). 33.Xu, C J.

> et al.

> > Phosphocreatine-dependent glutamate uptake by synaptic vesicles. A

> > comparison

> > with atp-dependent glutamate uptake. J. Bio. Chem. 271 (23), 13435-

> 13440

> > (1996).

> >

> > [0232] 34. Moriyama, Y., Tsai, H. L. & Futai, M. Energy-dependent

> > accumulation of neuron blockers causes selective inhibition of

> > neurotransmitter

> > uptake by brain synaptic vesicles. Arch. Biochem. Biophys. 305 (2),

> > 278-281

> > (1993).

> >

> > [0233] 35. Moriyama Y. & Futai, M. Presence of 5-hydroxytryptamine

> > (serotonin)

> > transport coupled with vacuolar-type H (+) -ATPase in

> neurosecretory

> > granules

> > from bovine posterior pituitary. J. Biol. Chem. 265 (16), 9165-9169

> > (1990).

> >

> > [0234] 36. Wolosker, H., de Souza, D. O. & de Meis, L. Regulation

> of

> > glutamate transport into synaptic vesicles by chloride and proton

> > gradient. J.

> > Biol. Chem. 271 (20), 11726-11731 (1996). 37. Brochier, G. &

> Morel, N.

> > The

> > same 15 kDa proteolipid subunit is a constituent of two different

> > proteins in

> > torpedo, the acetylcholine releasing protein mediatophore and the

> > vacuolar

> > H+ATPase. Neurochem. Int. 23 (6), 525-539 (1993).

> >

> > [0235] 38. Hayashi, M., Haga, M., Yatsushiro, S., Yamamoto, A. &

> > Moriyama,

> > Y. Vesicular monoamine transporter 1 is responsible for storage of

> >

> > [0236] 5-hydroxytryptamine in rat pinealocytes. J. Neurochem. 73

> (6),

> > 2538-2545 (1999).

> >

> > [0237] 39. Terland, O. & Flatmark, T. Drug-induced parkinsonism:

> > cinnarizine

> > and flunarizine are potent uncouplers of the vacuolar H+-ATPase in

> > catecholamine

> > storage vesicles. Neuropharmacology 38 (6), 879-882 (1999).

> >

> > [0238] 39. Miyamoto, J. K., Uezu, E., Jiang, P. J. & Miyamoto, A.

> T. H

> > (+) - ATPase and transport of DOPAC, HVA, and 5-HIAA in monoamine

> > neurons.

> > Physiol. Behav. 53 (1), 65-74 (1993). 40. Ramsay, A. M. & Rundle,

> A.

> > Clinical and biochemical findings in ten patients with benign

> rayalgic

> > encephalomyelitis. Postgrad. Med. J. 55 (654), 856-857 (1979).

> >

> > [0239] 41. Condon BR, Chaudhuri A, Condon BR, Gow JW, Hadley DN,

> Brennan

> > D,

> > " Proton magnetic resonance spectroscopy of basal ganglia in chronic

> > fatigue

> > syndrome " Neuroreport 14 (2), 225-228 (2003)

> >

> > [0240] 42. Chaudhuri A, Gow JW Behan PO, " Chronic fatigue syndrome

> and

> > systemic

> > viral infections: current evidence and recent advances. " in " Brain

> > Disease -

> > Therapeutic Strategies and Repair " Abramsky 0, Compston DA,

> A,

> > Said G.

> > 2002 pp 127-135.

> >

> > [0241] 43. Vernon, SD, Unger, ER, Dimulescu, IM, Rajeevan, M and

> Reeves,

> > WC.

> > Utility of the blood for gene expression profiling and biomarker

> > discovery in

> > chronic fatigue syndrome. Disease Markers 18, 193-199 (2002). 44.

> Klimas

> > NG,

> > Salvato FR, R, Fletcher MA. Immunological abnormalities in

> chronic

> > fatigue syndrome. J Clin Microbiol 1990; 28: 1403-10.

> >

> > [0242] 45. Lloyd AR, Wakefield D, Hickie I. Immunity and the

> > pathophysiology of

> > chronic fatigue syndrome. Ciba Found Symp 1993; 173: 176-87.

> >

> > [0243] 46. Barker E, Fuj imara SF, Fadem MB, Landay AL, Levy JA.

> > Immunological

> > abnormalities associated with chronic fatigue syndrome, Clin

> Infect Dis

> > 1994;

> > 18 (Suppl 1): S136-41.

> >

> > [0244] 47. Hassan IS, Bannister BA, Akbar A, Weir W, Bofill M. A

> study of

> > the

> > immunology of the chronic fatigue syndrome: correlation of

> immunologic

> > parameters to health dysfunction. Clin Immunol Immunopathol 1998;

> 87:

> > 60-7.

> >

> > [0245] 48. Vollmer-Conna U, Lloyd A, Hickie I, Wakefield D. Chronic

> > fatigue

> > syndrome: an immunological perspective. Aust NZ Psychiatry 1998;

> 32:

> > 523-7.

> > Table 6

> >

> > [0246]

> > <EMI ID=52.1>

> >

> > [0247]

> > <EMI ID=52.2>

> >

> > [0248]

> > <EMI ID=53.1>

> >

> > [0249]

> > <EMI ID=54.1>

> >

> > [0250]

> > <EMI ID=55.1>

> >

> > [0251]

> > <EMI ID=56.1>

> >

> > [0252]

> > <EMI ID=57.1>

> >

> > [0253]

> > <EMI ID=58.1>

> >

> > [0254]

> > <EMI ID=59.1>

> >

> > [0255]

> > <EMI ID=60.1>

> >

> > [0256]

> > <EMI ID=61.1>

> >

> > [0257]

> > <EMI ID=62.1>

> >

> > [0258]

> > <EMI ID=63.1>

> >

> > [0259]

> > <EMI ID=64.1>

> >

> > [0260]

> > <EMI ID=65.1>

> >

> > [0261]

> > <EMI ID=66.1>

> >

> > [0262]

> > <EMI ID=67.1>

> >

> > [0263]

> > <EMI ID=68.1>

> >

> > [0264]

> > <EMI ID=69.1>

> >

> > [0265]

> > <EMI ID=70.1>

> >

> > [0266]

> > <EMI ID=71.1>

> >

> > [0267]

> > <EMI ID=72.1>

> >

> > [0268]

> > <EMI ID=73.1>

> >

> > [0269]

> > <EMI ID=74.1>

> >

> > [0270]

> > <EMI ID=75.1>

> >

> > [0271]

> > <EMI ID=76.1>

> >

> > [0272]

> > <EMI ID=77.1>

> >

> > [0273]

> > <EMI ID=78.1>

> >

> > [0274]

> > <EMI ID=79.1>

> >

> > [0275]

> > <EMI ID=80.1>

> >

> > [0276]

> > <EMI ID=81.1>

> >

> > [0277]

> > <EMI ID=82.1>

> >

> > [0278]

> > <EMI ID=83.1>

> >

> > [0279]

> > <EMI ID=84.1>

> >

> > [0280]

> > <EMI ID=85.1>

> >

> > [0281]

> > <EMI ID=86.1>

> >

> > [0282]

> > <EMI ID=87.1>

> >

> > [0283]

> > <EMI ID=88.1>

> >

> > [0284]

> > <EMI ID=89.1>

> >

> > [0285]

> > <EMI ID=90.1>

> >

> > [0286]

> > <EMI ID=91.1>

> >

> > [0287]

> > <EMI ID=92.1>

> >

> > [0288]

> > <EMI ID=93.1>

> >

> > [0289]

> > <EMI ID=94.1>

> >

> > [0290]

> > <EMI ID=95.1>

> >

> > [0291]

> > <EMI ID=96.1>

> >

> > [0292]

> > <EMI ID=97.1>

> >

> > [0293]

> > <EMI ID=98.1>

> >

> > [0294]

> > <EMI ID=99.1>

> >

> > [0295]

> > <EMI ID=100.1>

> >

> > [0296]

> > <EMI ID=101.1>

> >

> > [0297]

> > <EMI ID=102.1>

> >

> > [0298]

> > <EMI ID=103.1>

> >

> > [0299]

> > <EMI ID=104.1>

> >

> > [0300]

> > <EMI ID=105.1>

> >

> > [0301]

> > <EMI ID=106.1>

> >

> > [0302]

> > <EMI ID=107.1>

> >

> > [0303]

> > <EMI ID=108.1>

> >

> > [0304]

> > <EMI ID=109.1>

> >

> > [0305]

> > <EMI ID=110.1>

> >

> > [0306]

> > <EMI ID=111.1>

> >

> > [0307]

> > <EMI ID=112.1>

> >

> > [0308]

> > <EMI ID=113.1>

> >

> > [0309]

> > <EMI ID=114.1>

> >

> > [0310]

> > <EMI ID=115.1>

> >

> > [0311]

> > <EMI ID=116.1>

> >

> > [0312]

> > <EMI ID=117.1>

> >

> > [0313]

> > <EMI ID=118.1>

> >

> > [0314]

> > <EMI ID=119.1>

> >

> > [0315]

> > <EMI ID=120.1>

> >

> > [0316]

> > <EMI ID=121.1>

> >

> > [0317]

> > <EMI ID=122.1>

> >

> > [0318]

> > <EMI ID=123.1>

> >

> > [0319]

> > <EMI ID=124.1>

> >

> > [0320]

> > <EMI ID=125.1>

> >

> > [0321]

> > <EMI ID=126.1>

> >

> > [0322]

> > <EMI ID=127.1>

> >

> > [0323]

> > <EMI ID=128.1>

> >

> > [0324]

> > <EMI ID=129.1>

> >

> > [0325]

> > <EMI ID=130.1>

> >

> > [0326]

> > <EMI ID=131.1>

> >

> > [0327]

> > <EMI ID=132.1>

> >

> > [0328]

> > <EMI ID=133.1>

> >

> > [0329]

> > <EMI ID=134.1>

> >

> > [0330]

> > <EMI ID=135.1>

> >

> > [0331]

> > <EMI ID=136.1>

> >

> > [0332]

> > <EMI ID=137.1>

> >

> > [0333]

> > <EMI ID=138.1>

> >

> > [0334]

> > <EMI ID=139.1>

> >

> > [0335]

> > <EMI ID=140.1>

> >

> > [0336]

> > <EMI ID=141.1>

> >

> > [0337]

> > <EMI ID=142.1>

> >

> > [0338]

> > <EMI ID=143.1>

> >

> > [0339]

> > <EMI ID=144.1>

> >

> > [0340]

> > <EMI ID=145.1>

> >

> > [0341]

> > <EMI ID=146.1>

> >

> > [0342]

> > <EMI ID=147.1>

> >

> > [0343]

> > <EMI ID=148.1>

> >

> >

> > Claims of WO2006082390

> > ----------------------

> >

> > Claims

> >

> > 1. A method for investigating whether a test subj ect is affected

> by

> > chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME),

> comprising

> > providing a biological sample from the subj ect and determining

> the level

> > in the sample of one or more biomarkers for CFS/ME, wherein the

> > biomarkers

> > comprise an expression product of at least one gene shown in Table

> 1.

> >

> > 2. A method according to claim 1, wherein the biomarkers comprise

> > expression products of a plurality of genes shown in Table 1.

> >

> > 3. A method according to claim 2 wherein the biomarkers comprise

> > expression products of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more

> > of the genes shown in Table 1.

> >

> > 4. A method according to any one of claims 1 to 3 wherein the

> biomarkers

> > comprise expression products of one or more of defensin [alpha]1,

> > haemoglobin [gamma], CXCR4, tubulin beta 1 and HLA DR[beta]4 genes.

> >

> > 5. A method according, to claim 4 wherein the biomarkers comprise

> > expression products of two, three, four or five of defensin [alpha]

> 1,

> > haemoglobin [gamma], CXCR4, tubulin beta 1 and HLA DR[beta]4 genes.

> >

> > 6. A method according to claim 4 or claim 5 wherein the biomarkers

> > comprise expression products of further hub genes of Table IA.

> >

> > 7. A method according to any one of claims 4 to 6 wherein the

> biomarkers

> > further comprise expression products of one or more of

> thrombospondin 1,

> > caspase 1 and/or IgG3.

> >

> > 8. A method according to any one of claims 1 to 7 wherein the

> biomarkers

> > comprise expression products of at least one gene from each of at

> least

> > two of Tables 2, 3, 4 and 5.

> >

> > 9. A method according to claim 8 wherein said at least one gene

> from each

> > said Table is a hub gene.

> >

> > 10. A method according to any one of the preceding claims wherein

> the

> > biomarkers comprise expression products of defensin cxl, CXCR4 and

> > lactotransferrin (LTF) genes.

> >

> > 11. A method according to claim 10 wherein the biomarkers comprise

> > expression products of defensin [alpha]4, integrin [alpha]2B,

> integrin

> > [beta]3, arginase 1, arginase 2, thrombospondin 1, membrane

> associated

> > protein 17 (MAP 17 ), Charcot Leyden Crystal Protein (CLC) and/or

> > chondroitin sulphate proteoglycan 2 (versican) genes.

> >

> > 12. A method according to any one of the preceding claims wherein

> the

> > biomarkers comprise an expression product of haemoglobin [gamma]

> (foetal

> > haemoglobin).

> >

> > 13. A method according to claim 12 wherein the biomarkers comprise

> > expression products of haemoglobin alpha 1, haemoglobin beta,

> haemoglobin

> > delta, prostaglandin-endoperoxide synthase 1 and/or

> > prostaglandin-endoperoxide synthase 2 genes.

> >

> > 14. A method according to any one of the preceding claims wherein

> the

> > biomarkers comprise an expression product of serine/threonine

> kinase 17B

> > (STK17B).

> >

> > 15. A method according to claim 14 wherein the biomarkers comprise

> > expression products of caspase 1, dynamin 1-like, and/or

> phosphatidyl

> > serine binding protein genes.

> >

> > 16. A method according to any one of the preceding claims wherein

> the

> > biomarkers comprise an expression product of HLA-DR[beta]4.

> >

> > 17. A method according to claim 16 wherein the biomarkers comprise

> > expression products of HLA-DQ[beta]l and/or immunoglobulin heavy

> chain

> > [gamma]3 genes.

> >

> > 18. A method according to any one of the preceding claims wherein

> the

> > biomarkers comprise an expression product of one or more further

> genes

> > from Table 6.

> >

> > 19. A method according to any one of the preceding claims wherein

> the

> > biomarkers comprise an expression product of prostaglandin D2

> synthase.

> >

> > 20. A method for classifying a subject affected by CFS/ME, the

> method

> > comprising providing a biological sample from the subj ect and

> > determining

> > the level in the sample of one or more biomarkers for CFS/ME,

> wherein the

> > biomarkers comprise expression products of at least one gene shown

> in one

> > or more of Tables 2 to 5.

> >

> > 21. A method according to claim 20 wherein the biomarkers comprise

> > expression products of a hub gene from one of Tables 2, 3, 4 or 5.

> >

> > 22. A method according to claim 21 wherein the biomarkers comprise

> > expression products of hub genes from each of two, three or all of

> Tables

> > 2, 3, 4 or 5.

> >

> > 23. A method according to any one of claims 20 to 22 further

> comprising

> > determining network genes from one, two, three or all of Tables 2,

> 3, 4

> > and 5.

> >

> > 24. A method according to any one of claims 20 to 23 wherein the

> > biomarkers comprise expression products of defensin [alpha]1,

> CXCR4 and

> > lactotransferrin (LTF) genes.

> >

> > 25. A method according to claim 24 wherein the biomarkers comprise

> > expression products of defensin [alpha]4, integrin [alpha]2B,

> integrin

> > [beta]3, arginase 1, arg+-nase 2, thrombospondin 1, membrane

> associated

> > protein 17 (MAP 17 ), Charcot Leyden Crystal Protein (CLC) and/or

> > chondroitin sulphate proteoglycan 2 (versican) genes.

> >

> > 26. A method according to any one of claims 20 to 25 wherein the

> > biomarkers comprise an expression product of haemoglobin Y (

> foetal

> > haemoglobin).

> >

> > 27. A method according to claim 26 wherein the biomarkers comprise

> > expression products of haemoglobin alpha 1, haemoglobin beta,

> haemoglobin

> > delta, prostaglandin-endoperoxide synthase 1 and/or

> > prostaglandin-endoperoxide synthase 2 genes.

> >

> > 28. A method according to any one of claims 20 to 27 wherein the

> > biomarkers comprise an expression product of serine/threonine

> kinase 17B

> > (STK17B).

> >

> > 29. A method according to claim 28 wherein the biomarkers comprise

> > expression products of caspase 1, dynamin 1-like, and/or

> phosphatidyl

> > serine binding protein genes.

> >

> > 30. A method according to any one of claims 20 to 28 wherein the

> > biomarkers comprise an expression product of HLA-DR[beta]4.

> >

> > 31. A method according to claim 30 wherein the biomarkers comprise

> > expression products of HLA-DQ[beta]l and/or immunoglobulin heavy

> chain

> > [gamma]3 genes.

> >

> > 32. A method according to any one of claims 20 to 31 comprising

> > classifying the subject as being affected by one or more of

> oxidative

> > stress, excessive apoptosis, and immunological dysregulation (MHC

> I to II

> > shift).

> >

> > 33. A method of determining whether an individual affected by

> CFS/ME is

> > suitable for treatment with an anti-oxidant, minocycline or a

> > corticosteroid, comprising determining whether the individual is

> affected

> > by one or more of oxidative stress, excessive apoptosis, and

> > immunological

> > dysregulation by a method according to any one of claims 20 to 24,

> and

> > prescribing a suitable treatment depending on the outcome.

> >

> > 34. A method of creating a library of expression profiles for use

> in

> > determining whether an individual is affected by CFS/ME, the

> method

> > comprising

> > (a) providing biological samples from a plurality of individuals

> affected

> > by CFS/ME, and determining the level in each sample of one or more

> > biomarkers for CFS/ME to create a plurality of expression profiles

> from

> > affected individuals;

> > ( providing biological samples from a plurality of individuals

> not

> > affected by CFS/ME, and determining the level in the sample of

> said one

> > or

> > more biomarkers for CFS/ME to create a plurality of expression

> profiles

> > from unaffected individuals;

> > wherein the biomarkers comprise expression products of one or more

> genes

> > shown in Table 1.

> >

> > 35. A method according to claim 34 comprising the step of

> retrievably

> > storing each of the expression profiles on a computer data

> carrier, in

> > order to create a database of expression profiles for both

> affected and

> > unaffected individuals.

> >

> > 36. A method of determining the efficacy of a treatment for CFS/ME

> > comprising the steps of:

> > (a) providing a biological sample from a subj ect affected by

> CFS/ME who

> > has been subj ected to said treatment, ( determining the level

> in said

> > sample of one or more biomarkers for CFS/ME to create an

> expression

> > profile for said subj ect, and

> > [c) comparing said expression profile with

> > i) a comparable expression profile obtained from said test subject

> before

> > initiation of said treatment, and/or

> > ii) a comparable expression profile obtained from said test

> subject at an

> > earlier stage of said treatment, and/or

> > iii) a comparable expression profile characteristic of a subj ect

> who is

> > unaffected by CFS/ME,

> > wherein the one or more biomarkers for CFS/ME comprise expression

> > products

> > of one or more genes shown in Tables 1 to 5.

> >

> > 37. A method according to any one of the preceding claims wherein

> the

> > biological sample is a blood sample or comprises peripheral blood

> > mononuclear cells isolated from a blood sample.

> >

> > 38. A method according to any one of the preceding claims wherein

> the

> > expression level of each of the biomarkers is determined by

> contacting

> > the

> > sample with a binding agent capable of binding specifically to

> said

> > expression product of the corresponding gene.

> >

> > 39. A method according to claim 38 wherein the expression product

> is a

> > nucleic acid.

> >

> > 40. A method according to claim 39 wherein the binding agent is a

> nucleic

> > acid probe or primer.

> >

> > 41. A method according to claim 39 or claim 40 wherein the

> expression

> > product is mRNA, pre-r[alpha]RNA or cDNA.

> >

> > 42. A method according to any one of claims 39 to 41 wherein the

> > expression level of the biomarker is determined by means of

> Northern

> > blot,

> > in situ hybridisation, RT-PCR, Southern blot or microarray assay.

> >

> > 43. A method according to claim 38 wherein the expression product

> is a

> > polypeptide.

> >

> > 44. A method according to claim 43 wherein the binding agent is a

> ligand

> > or receptor for said polypeptide, or an antibody or fragment

> thereof

> > specific for said polypeptide

> >

> > 45. A method according to claim 43 or claim 44 wherein the

> expression

> > level of the biomarker is determined by Western blot, ELISA or

> microarray

> > assay.

> >

> > 46. A method according to any one of the preceding claims further

> > comprising the step of determining the level, in the biological

> sample,

> > or in a further biological sample from the subj ect, of the

> peptide QYNAD.

> >

> > 47. A method according to claim 46 wherein the further biological

> sample

> > is a blood or serum sample.

> >

> > 48. A method according to claim 46 wherein the further biological

> sample

> > is cerebrospinal fluid.

> >

> > 49. A kit for use in a method according to any one of the preceding

> > claims,

> > the kit comprising a plurality of binding agents, each capable of

> > binding specifically and individually to an expression product of

> one of

> > the genes of Table 1, or the peptide QYNAD.

> >

> > 50. A kit according to claim 49 comprising binding agents specific

> for

> > expression products of two or more genes of Tables 1, or at least

> one

> > gene of Table 1 and the peptide QYNAD.

> >

> > 51. A kit according to claim 50 comprising binding agents capable

> of

> > binding specifically and individually to expression products of 2,

> 3, 4,

> > 5, 6, 7, 8, 9, 10, 15, 20 or more of the genes of Table 1 and

> > optionally the peptide QYNAD.

> >

> > 52. A kit according to any one of claims 49 to 51 wherein the

> binding

> > agents are immobilised on one or more solid supports.

> >

> > 53. A kit according to any one of claims 49 to 52 further

> comprising one

> > or more binding agents capable of binding specifically to an

> expression

> > product of a control gene which is substantially not

> differentially

> > expressed between individuals affected and whose expression level

> is

> > substantially unaffected by CFS/ME.

> >

> > 54. A kit according to claim 53 wherein the control gene is

> > glyceraldehyde

> > phosphate dehydrogenase (GAPDH), [beta]-actin, or abl (ableson

> tyrosine

> > kinase).

> >

> > 55. A kit according to any one of claims 49 to 54 comprising

> binding

> > agents for expression products of less than 1000 different genes,

> less

> > than 500 different genes, less than 100, less than 50, less than

> 40,

> > less than 30, less than 20, or less than 10 different genes.

> >

> > 56. A kit according to any one of claims 49 to 55 wherein the

> expression

> > product is a nucleic acid and the binding agent is a nucleic acid

> probe

> > or

> > primer.

> >

> > 57. A kit according to claim 56 wherein the expression product is

> mRNA,

> > pre-mRNA or cDNA.

> >

> > 58. A kit according to any one of claims 49 to 55 wherein the

> expression

> > product is a polypeptide and the binding agent is a ligand or

> receptor

> > for

> > said polypeptide, or an antibody or fragment thereof specific for

> said

> > polypeptide

> >

> > 59. An expression profile database, comprising a plurality of

> expression

> > profiles of biomarkers for CFS/ME from affected and unaffected

> > individuals

> > , wherein the biomarkers comprise expression products of one or

> more

> > genes

> > shown in Table 1.

> >

> > 60. A method of treating CFS/ME in an individual suffering

> therefrom,

> > comprising administering an effective amount of an antioxidant.

> >

> > 61. A method according to claim 60 further comprising

> administering

> > therapeutically effective amounts of a corticosteroid and/or

> minocyline.

> >

> > 62. Use of an anti-oxidant in the preparation of a medicament for

> the

> > treatment of CFS/ME.

> >

> > 63. Use according to claim 62 wherein the medicament is formulated

> for

> > administration in conjunction with a corticosteroid and/or

> minocycline,

> > or

> > comprises a corticosteroid and/or minocycline.

> >

> > 64. A method according to claim 60 or claim 61, or use according

> to claim

> > 62 or claim 63, wherein the anti-oxidant is coenzyme Q10 or an

> inhibitor

> > of a cyclooxygenase (COX) enzyme such as celecoxib (4- [5-

> > (4-methylphenyl) -3- (trifluoromethyl ) -1H-pyrazol-1-yl]

> > benzenesulfonamide).

> >

> > 65. A method of treating CFS/ME in an individual suffering

> therefrom,

> > comprising administering an effective amount of minocycline.

> >

> > 66. A method according to claim 65 further comprising

> administering a

> > therapeutically effective amount of a corticosteroid and/or an

> > anti-oxidant.

> >

> > 67. Use of minocycline in the preparation of a medicament for the

> > treatment of CFS/ME.

> >

> > 68. Use according to claim 67 wherein the medicament is formulated

> for

> > administration in conjunction with a corticosteroid and/or an

> > anti-oxidant, or comprises a corticosteroid and/or an anti-oxidant.

> >

> > 69. A method of treating CFS/ME in an individual suffering

> therefrom,

> > comprising administering an effective amount of a corticosteroid.

> >

> > 70. A method according to claim 69 further comprising

> administering a

> > therapeutically effective amount of minocycline and/or an anti-

> oxidant.

> >

> > 71. Use of a corticosteroid in the preparation of a medicament for

> the

> > treatment of CFS/ME.

> >

> > 72. Use according to claim 71 wherein the medicament is formulated

> for

> > administration in conjunction with minocycline and/or an

> antioxidant, or

> > comprises minocycline and/or an anti-oxidant.

> >

> > 73. A method according to claim 69 or claim 70, or use according

> to claim

> > 71 or claim 72, wherein the corticosteroid is hydrocortisone,

> > dexamethasone or prednisone.

> >

> > 74. A pharmaceutical composition comprising a therapeutically

> effective

> > amount of minocycline in combination with a therapeutically

> effective

> > amount of a corticosteroid and/or an anti- oxidant, and a

> > pharmaceutically

> > acceptable carrier..

> >

> > 75. A pharmaceutical composition comprising a therapeutically

> effective

> > amount of a corticosteroid in combination with a therapeutically

> > effective

> > amount of minocycline and/or an anti- oxidant, and a

> pharmaceutically

> > acceptable carrier.

> >

> > 76. A pharmaceutical composition comprising a therapeutically

> effective

> > amount of an anti-oxidant in combination with a therapeutically

> effective

> > amount of a corticosteroid and/or minocycline, and a

> pharmaceutically

> > acceptable carrier.

> >

> >

> > Mosaics of WO2006082390

> > -----------------------

> >

> > [see European Patent Office Document

> >

> > http://v3.espacenet.com/textdraw?

> DB=EPODOC & IDX=WO2006082390 & F=0 & QPN=WO2006082390

> >

> >

> > Original Document of WO2006082390

> > ---------------------------------

> >

> > [see European Patent Office Document

> >

> > http://v3.espacenet.com/origdoc?

> DB=EPODOC & IDX=WO2006082390 & F=0 & QPN=WO2006082390

> >

> >

> > INPADOC legal status of WO2006082390

> > ------------------------------------

> >

> > No legal data found.

> >

> > --------

> > © 2006 European Patent Office

> >

>

[Guest guest]

>

> Has anyone had experience of using the drugs Dr Gow reccommends ?

> Corticosteroid and minocycline ?

>

>

>

Hi

I have been on Prednisolone for over 3 years and previous to that

was on hydrocortisone for 6 months (together with thyroid meds).

They literally gave me some quality of life but I haven't been able

to stop them for any period of time because my adrenals or HPA axis

is so weak. I have no side effects at all. The only problem I have

encountered is that because they help you to feel better I quite

often end up doing too much physical stuff and then I can get some

horrendous migraines. However I do recover very quickly.

I have never taken more than the equivalent of 20 mg h/c or 5mg

Prednisolone and I am interested that Dr Gow talked

about " therapeutic doses " which I think might be more in my case,

I'm not sure.

I have also taken antibiotics for quite long periods but I have

problems with some of them cos they block the effect of the Pred

(same detoxification pathway). Unfortunately I seemed to be

allergic to Mino but have always done pretty well on Doxycycline

which is quite similar. They do give me bad yeast issues though.

My problem is I have persistent viruses in my DNA and I haven't yet

found a way around that. Not sure what Dr Gow said about that will

have to have another look.

Pam

[Guest guest]

Hello everybody. I live in Spain and went some months ago to a

famous spanish reumatologist (dr FERRAN) and asked him abt the work

of dr Gow. He answered me litterally : " I know ver well the work of

Dr. Gow, but he has a basic problema that, in my opinión have

all the authors excepted a few: they simply mix in their study

patients affected with FM, and therefore their markers are a mix

between the real from CFS (we also have some of these) and others

that are not.

In the actual phase the important is that the patients are well

segmented in the studies in order not to confound the results and,

when we manage that, a scoring of all the SNP's, that mostly appear

in the studies, must been done.

As the base is wrong, the resto also will be wrong. But that is only

my opinión " .

Whta is your opinion abt that? I'm talking abt a doctor who is also

making a genetic study abt CFS/FM: the biggest one made ever in

Spain. He is one of the most famous doctors in Spain abt FM/CFS and

is very nice with the patients

greetings

[Guest guest]

,

You might want to ask your rheumatologist how he distinguishes

between patients with cfs or fms.

a Carnes

>

> Hello everybody. I live in Spain and went some months ago to a

> famous spanish reumatologist (dr FERRAN) and asked him abt the work

> of dr Gow. He answered me litterally : " I know ver well the work of

> Dr. Gow, but he has a basic problema that, in my opinión have

> all the authors excepted a few: they simply mix in their study

> patients affected with FM, and therefore their markers are a mix

> between the real from CFS (we also have some of these) and others

> that are not.

> In the actual phase the important is that the patients are well

> segmented in the studies in order not to confound the results and,

> when we manage that, a scoring of all the SNP's, that mostly appear

> in the studies, must been done.

> As the base is wrong, the resto also will be wrong. But that is

only

> my opinión " .

>

> Whta is your opinion abt that? I'm talking abt a doctor who is also

> making a genetic study abt CFS/FM: the biggest one made ever in

> Spain. He is one of the most famous doctors in Spain abt FM/CFS and

> is very nice with the patients

> greetings

>

>

[Guest guest]

& all,

Thanks for bringing this to our attention regarding the possibility of Dr. Gow

mixing

ME/CFS

with FMS in his study. That's something that should be addressed if that is

true. I

wonder how

we could check out this information.

wrote in part:

" Dr. Gow, but he has a basic problema that, in my opinión have

all the authors excepted a few: they simply mix in their study

patients affected with FM, and therefore their markers are a mix

between the real from CFS (we also have some of these) and others

that are not. "

Du Pre

Poetry website: http://www.angelfire.com/poetry/soareagle/index.html

" By words the mind is winged. " Aristophanes

Website for National Alliance for Myalgic Encephalomyelitis:

http://www.name-us.org

[Guest guest]

Hi and ,

Well I've listened to a DVD of a talk Gow gave last year on his gene

expression work and he was all to well aware of getting the correct

patients with CFS/ME.

I can't remember the figures exactly but he said they looked at all

the patients that had been refered to the hospital by GPs with CFS/ME

and they found that a large percentage of them didn't have the illness

but had other illnesses so they were being quite specific in how they

chose the people for the gene expression testing.

He did say that when they expanded the numbers of people being tested

for further research they would have to be careful that the people

being refered did genuinely have CFS as diferent doctors follow

different definitions, as we know all to well.

He also said that Professor Simon Wessely had suggested that he

collaborated with his colleague Professot Sharpe who would supply Gow

with CFS patients from his CFS clinic. I am glad to say, that at that

point last year, Gow wasn't going to take up that offer and I hope he

hasn't done so now because Wessely and Sharpes' definition of CFS is

that it is a somatoform disorder, due to us wrongly thinking we are

ill therefore not CFS/ME as defined by say the Canadian Diagnostic

Criteria.

BW,

Sheila

>

> & all,

> Thanks for bringing this to our attention regarding the possibility

of Dr. Gow mixing

> ME/CFS

> with FMS in his study. That's something that should be addressed if

that is true. I

> wonder how

> we could check out this information.

>

> wrote in part:

> " Dr. Gow, but he has a basic problema that, in my opinión have

> all the authors excepted a few: they simply mix in their study

> patients affected with FM, and therefore their markers are a mix

> between the real from CFS (we also have some of these) and others

> that are not. "

>

> Du Pre

> Poetry website: http://www.angelfire.com/poetry/soareagle/index.html

> " By words the mind is winged. " Aristophanes

> Website for National Alliance for Myalgic Encephalomyelitis:

http://www.name-us.org

>

>

>

[Guest guest]

Hi , Sheila and all,

The patent refers to a three-fold approach depending on specific gene

expression... I think.

1. An antioxidant (they list Co-Q-10 and -2 inhibitors such as Celecoxib as

examples)

2. Minoclyine (which they say inhibits Caspase 1)

3. A coristeroid (they list hydocortisone, dexamethasone and prednisone as

examples)

From this list I've tried.

1. Co-Q-10 with no obvious help.

2. Not tried Minoclyine

3. Hydrocortisone with no obvious help, Dexamethasone - makes me feel toxic and

over stimulated

and Prednisone - which really suits me but I have not tried for a long period of

time.

Kindest regards,

Annette

___________________________________________________________

Try the all-new . " The New Version is radically easier to use " – The

Wall Street Journal

http://uk.docs./nowyoucan.html

[Guest guest]

How does anyone distinguish cfs from fms? Are we using the Canadian

guidelines? Lots of patients are labeled cfs by doctors who never

read any guidelines let alone the Canadian ones. Did Gow pull from

these patients and these doctors? Did the rheumatologist in Spain use

particular guidelines to determine who has what? Did Gow use the DCD

list or the Canadian guidelines?

And then there is borrelia which some here do not want to discuss.

Let me just repeat that three of us in my family have the same

infection, borrelia. I am the only one who looks like cfs and has

disablity based on a cfs diagnosis. Other than the first year I got

diagnosed with cfs and had a disease that looked like chronic mono I

would have gotten a fibromyalgia label any other year of my life

since 1972, had there been such a label. The other two members of my

family don't look anything like cfs or fms, yet they have the same

disease as I do.

What am I missing here??? Who knows what Gow is testing? Probably no

one, and not Gow either. We simply are unable to correctly label any

of us in the world at this time with existing tests.

a Carnes

>

> Hi and ,

>

> Well I've listened to a DVD of a talk Gow gave last year on his gene

> expression work and he was all to well aware of getting the correct

> patients with CFS/ME.

>

> I can't remember the figures exactly but he said they looked at all

> the patients that had been refered to the hospital by GPs with

CFS/ME

> and they found that a large percentage of them didn't have the

illness

> but had other illnesses so they were being quite specific in how

they

> chose the people for the gene expression testing.

>

> He did say that when they expanded the numbers of people being

tested

> for further research they would have to be careful that the people

> being refered did genuinely have CFS as diferent doctors follow

> different definitions, as we know all to well.

>

> He also said that Professor Simon Wessely had suggested that he

> collaborated with his colleague Professot Sharpe who would supply

Gow

> with CFS patients from his CFS clinic. I am glad to say, that at

that

> point last year, Gow wasn't going to take up that offer and I hope

he

> hasn't done so now because Wessely and Sharpes' definition of CFS is

> that it is a somatoform disorder, due to us wrongly thinking we are

> ill therefore not CFS/ME as defined by say the Canadian Diagnostic

> Criteria.

>

> BW,

> Sheila

>

>

>

> >

> > & all,

> > Thanks for bringing this to our attention regarding the

possibility

> of Dr. Gow mixing

> > ME/CFS

> > with FMS in his study. That's something that should be addressed

if

> that is true. I

> > wonder how

> > we could check out this information.

> >

> > wrote in part:

> > " Dr. Gow, but he has a basic problema that, in my opinión

have

> > all the authors excepted a few: they simply mix in their study

> > patients affected with FM, and therefore their markers are a mix

> > between the real from CFS (we also have some of these) and others

> > that are not. "

> >

> > Du Pre

> > Poetry website:

http://www.angelfire.com/poetry/soareagle/index.html

> > " By words the mind is winged. " Aristophanes

> > Website for National Alliance for Myalgic Encephalomyelitis:

> http://www.name-us.org

> >

> >

> >

[Guest guest]

I want to draw people's attention to the segment of the paper which

describes the reasoning behind the use of minocycline (below), as I've

noticed a couple of the replies here mention its use as an antibiotic,

or in the same breath as other antibiotics.

[0088] Patients showing evidence of abnormal apoptosis may be treated

with minocyline, which is an inhibitor of caspase 1, shown here to be

upregulated in individuals affected by CFS/ME. Minocyline has

previously been suggested for treatment of various neurological

disorders including stroke, multiple sclerosis,spinal cord injury,

Parkinson' s disease, Huntington' s disease and amylotrophic lateral

sclerosis - see Wee Young et al., Lancet Neurology,2004,

744-751 for review. Thus the invention provides a method of treating

CFS/MEin an individual suffering therefrom, comprising administering

an effective amount of minocycline.

>

>

> Interesting. Their protocol would basically treat inflammation,

infection (bacterial) and oxidative stress...which is what many

protocols already address.

>

> Good to know we're on the right track.

>

> Thanks for posting.

> Louella

>

>