Patent Application: US-81529006-A

Abstract:
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 α1 , haemoglobin γ , 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:
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 &# 39 ; 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 . 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 . 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 . 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 . 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 ). 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 . 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 ). 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 . 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 watson - 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 . 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 . 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 . 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 . 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 . in alternative embodiments , the analyte can be tagged before applying it to the support comprising the binding agent . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ch1 domains ; ( ii ) the fd fragment consisting of the vh and ch1 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 . 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 . 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 . physiological saline solution , dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included . for intravenous , cutaneous or subcutaneous injection , or injection 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 injection , ringer &# 39 ; s injection , lactated ringer &# 39 ; s injection . preservatives , stabilisers , buffers , antioxidants and / or other additives may be included , as required . 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 time - 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 &# 39 ; s pharmaceutical sciences , 20th edition , 2000 , pub . lippincott , williams & amp ; wilkins . 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 . a composition may be administered alone or in combination with other treatments , either simultaneously or sequentially dependent upon the condition to be treated . a group at the university of ulm , 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 . 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 ulm 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 . when the code was broken , the results showed that the disease control group had levels of the pentapeptide which were 2 . 3 × those of the normal controls ( similar to the published data ) and the cfs samples had levels which were 3 × higher than the healthy controls . 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 . 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 . 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 fig1 , 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 . 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 . 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 subjects , giving an insight into the biochemical pathways which are involved in this disorder . 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 . 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 . 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 )). 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 . further genes , including prostaglandin d2 synthase and t - cell receptors alpha , beta , gamma and delta are found to be down - regulated 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 . 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 ii receptors and downregulation of t - cell receptor - α , 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 . functional changes produced by altered gene regulation may explain the mechanism of fatigue and offer a rational basis for targeted pharmacotherapy in cfs . 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 . 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 . fig2 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 . fig2 shows rt - pcr analysis of defensin alpha 1 , fig3 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 . fig6 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 . 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 . 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 . 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 . bailey et al 27 have shown a relationship between potassium depletion and up - regulation of h +- atpase . 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 involved 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 . 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 . patients with cfs were diagnosed with reference to the 1994 fukuda definition . all seven patients were male , aged between 18 and 54 years ( mean 3 6 ), and were not on medication . healthy control subjects 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 . venous blood samples were drawn from patients who fulfilled the holmes and fukuda criteria for me / cfs , and from healthy individuals . the procedure for isolating pbmc was started immediately and finished within 2 h 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 ° c . at 500 g 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 ( 155 mm nh4cl , 10 mm nahco3 , ph7 . 4 , 0 . 1 mm edta ). the pbmc were centrifuged once more ( 20 ° c ., 500 g , 10 min ). the pbmc were then aliquoted into microtubes equivalent to 5 × 105 cells per tube , centrifuged and stored as dry pellets at − 80 ° c . pbmc pellets were resuspended in sample reducing buffer ( 1 ml glycerol , 0 . 5 ml β - mercaptoethanol , 3 ml 10 % sds , 1 . 25 ml 1m tris - hcl ph 6 . 7 ), boiled for 5 min . the lysates were loaded onto a 10 % page gel , each track equivalent to 2 × 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 defensin1 - 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 ) ( jackson immunoresearch laboratories pa usa ). the reactions were detected using sigma fast bcip / nbt ). 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 . total rna was isolated from peripheral blood mononuclear cells ( pbmc ) using the promega rnagents total rna isolation system . 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 , 25 mm ; n - laurolyl sarcosine , 0 . 5 %; 2 - mercaptoethanol , 0 . 1m , in distilled water adjusted to ph 7 . 0 ). to homogenate ( 1 ml ), sodium acetate ( 100 μl , 2m , ph 4 . 0 ), citrate - buffered ( 0 . 1m , ph 4 . 3 ) phenol ( 1 ml ) and chloroform : isoamyl alcohol ( 49 : 1 , 200 μl ) 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 , 000 g , 20 minutes , 4 ° c .) and the upper aqueous layer pippetted into to a fresh tube . after addition of ice cold isopropanol ( 1 ml ), rna precipitated from the mixture during a thirty minute period on dry ice . the mixtures were then centrifuged ( 12 , 000 g , 20 minutes , 4 ° c .) and the supernatant discarded . the pellet was dissolved in denaturing solution ( 300 μl ) and transferred to a microcentrifuge tube ( 1 . 5 ml , axygen ). ethanol ( absolute , 600 μl ) was added to each microcentrifuge tube , samples were incubated on dry ice for 30 minutes , and then centrifuged ( 11600 g , 20 minutes , 4 ° 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 μl ) and stored at − 70 ° c . until required . rna solution ( prepared as described above , 2 μl ) was added to distilled water ( 98 μl ) to produce a 1 : 50 dilution . the optical density of the sample was read at 260 nm and 280 nm using a 50 μl ultraviolet cuvet . the absorbance ratio 260 / 280 nm measured at these wavelengths indicates the purity of the rna . the absolute concentration of rna was estimated using the following equation : to examine the quality , 5 μl of rna was added to 5 μl of the gel marker orange g ( 1 % orange g dye in 50 % glycerol , 50 % 2 × tbe ). this was heated to 70 ° c . for 3 minutes , then electrophoresed through a horizontal 1 % agarose gel in 1 × tbe ( 10 × 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 ( 320 nm ) ultraviolet light . two micrograms of rna were added to 1 μl oligo ( dt ) 12 - 18 ( 500 μg / ml , roche ) and the volume made up to 11 μl with sterile distilled water . this was incubated ( 70 ° 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 μl of 5 × first strand buffer , 2 μl of 0 . 1m dtt , 1 μl of 10 mm dntp mix ( 10 mm each datp , dgtp , dctp , dttp ; amersham pharmacia biotech ), 1 μl of sterile distilled water and 1 μl of superscript ii ( gibco - brl ® life technologies ) were added . this was incubated first at 50 ° c . for 1 hr , then 70 ° c . for 15 minutes to inactivate the reaction . a negative cdna control ( 2 μl of distilled water ) was included in each cdna synthesis to confirm that the reaction mix was not contaminated . to 2 μl of cdna , 10 μl of 10 × magnesium - free buffer , 10 μl of 2 . 5 mm dntps ( 2 . 5 mm each datp , dgtp , dctp , dttp ), 6 μl mgcl 2 ( 25 mm ) and 1 μl each of the appropriate 5 ′ and 3 ′ primers ( 0 . 5 μg / μl ) were added . the volume was made up to 99 . 8 μl using sterile distilled water and 0 . 2 μl of taq dna polymerase ( promega ) was added . the pcr reaction ( 35 cycles ) was carried out on a techne genius thermocycler . primers used to amplify vatpase were 5 ′- ctc gtg acc tgt tac tgc tg - 3 ′ and 5 ′- aag taa cca agt cca ctc 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 ′. thirty microlitres of pcr product was added to 5 μl of orange g and electrophoresed through a horizontal 2 - 3 % agarose gel in 1 × tbe at 100 volts , until the dye front had migrated a minimum of 10 cm . on each gel , 3 μl of a 123 base pair dna ladder in 5 μl 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 ( 320 nm ) ultraviolet light . 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 . 1 . chaudhuri a , behan p o . neurological dysfunction in chronic fatigue syndrome . j chr fatigue synd 2000 ; 6 ( 3 / 4 ): 51 - 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