WO2008156418A1 - A method for screening or diagnosis of postpolio syndrome and fibromyalgia - Google Patents
A method for screening or diagnosis of postpolio syndrome and fibromyalgia Download PDFInfo
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- WO2008156418A1 WO2008156418A1 PCT/SE2008/050721 SE2008050721W WO2008156418A1 WO 2008156418 A1 WO2008156418 A1 WO 2008156418A1 SE 2008050721 W SE2008050721 W SE 2008050721W WO 2008156418 A1 WO2008156418 A1 WO 2008156418A1
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/105—Poliovirus
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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Definitions
- the present invention relates to the identification of polypeptides, proteins and protein isoforms that are associated with Postpolio Syndrome (PPS), and to their use for e.g., clinical screening, diagnosis, prognosis, therapy and prophylaxis, as well as for drug screening and drug development.
- PPS Postpolio Syndrome
- the present invention provides methods and compositions for clinical screening, diagnosis and treatment of PPS, for monitoring the effectiveness of PPS treatment, for selecting participants in clinical trials, for identifying patients most likely to respond to a particular therapeutic treatment and for screening and development of drugs for treatment of PPS.
- the Post-Polio Syndrome is a condition appearing several decades after the acute poliomyelitis infection. Survivors of acute paralytic disease often develop new or increased muscle weakness, fatigue and pain from the musculoskeletal system (Halstead LS, Rossi CD, Orthopedics, 1985: 845-50). Even other symptoms as breathing difficulties, swallowing problems and other brain stem symptoms may appear.
- PPS post-polio syndrome
- the OND group consisted of patients with pain syndromes, vertigo, sensory symptoms, and fatigue. One patient suffered from psychosis. Ten healthy controls were included.
- the CSF of persons with PPS was characterized by differential expression of 5 distinct proteins: gelsolin, hemopexin, peptidylglycine alpha-hydroxylating monooxygenase (PAM), glutathione synthetase (GSHB), and kallekrein 6. These proteins are involved in apoptosis and/or inflammation.
- the data provide strong evidence that the clinical deteroriation during PPS is due to an active inflammatory neurodegeneration, of potential therapeutic relevance.
- the data set the stage for diagnostic and/or prognostic evaluation of these proteins.
- Figure 1 Data analysis strategy.
- the model optimisation was performed on a set of 1499 spots that included 36 OND/HC and 9 PPS samples. In the optimization procedure, this set of samples was randomly divided into training and test sets, repeatedly, to obtain an estimate of the prediction performance. From this analysis, the spots were ranked by importance in the model, and the top 13 spots that were identified using mass spectrometry, were chosen to constitute the final model.
- the samples used to train the model were the 36+9 samples used in the model optimization, and this model was tested on a new independent set of samples, 6 PPS and 7 OND samples.
- FIG. 1 2D-gel separation of CSF proteins from one PPS patient.
- the positions of the top- 13 identified protein spots are shown, hemopexin, gelsolin, kallikrein-6, peptidylglycine alpha-hydroxylating monooxygenase (PAM) and glutathione synthetase (GSHB).
- PAM peptidylglycine alpha-hydroxylating monooxygenase
- GSHB glutathione synthetase
- the right panel show zoom-in areas of identified proteins and SSP number. Mass spectrometry data for the top- 13 proteins are shown in Table 1.
- Figure 3 Box-plots showing expression levels in ppm for 43 OND/HC and 15 samples. The boxes are defined by the 25th and 75th percentile, thus 50 percent of the data is represented within each box. Upper and lower adjacent values, are represented by whiskers (defined by SpotfireTM). The median is represented by blue arrows, the mean is represented by black arrows.
- the present invention originates from a study that originates from observations in another study. All patient samples were first analysed in a study focusing on unbiased identification of biomarkers in multiple sclerosis (manuscript in preparation). In that study, PPS patient samples were included to serve as a reference disease material. However, results from the PPS group were very interesting and deserves to be presented separately and with the aims given above.
- a method for screening or diagnosis of Postpolio Syndrome (PPS) in a subject for determining the stage or severity of PPS in a subject, for identifying a subject at risk of developing PPS, or for monitoring the effect of therapy administered to a subject having PPS, said method comprising:
- said array comprises one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
- PPS Features PPS Features
- a method for screening or diagnosis of Postpolio Syndrome (PPS) in a subject for determining the stage or severity of PPS in a subject, for identifying a subject at risk of developing PPS, or for monitoring the effect of therapy administered to a subject having PPS, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following proteins: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB.
- PPS Postpolio Syndrome
- said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
- PPS Features PPS Features
- said body fluid is cerebrospinal fluid (CSF).
- CSF cerebrospinal fluid
- said body fluid is plasma
- said body fluid is serum.
- said body fluid is urine
- an antibody capable of immunospecific binding to a Postpolio Syndrome Feature (PPSF).
- PPSF Postpolio Syndrome Feature
- said antibody is a monoclonal antibody.
- said antibody is a chimeric antibody.
- said antibody is a bispecific antibody.
- said antibody is a humanised antibody.
- a kit comprising one or more antibodies as defined above and/or one or more PPSF as defined above, other reagents and instructions for use.
- said kit is for use in the screening or diagnosis of Postpolio Syndrome in a subject, for determining the stage or severity of Postpolio
- said kit comprising a plurality of antibodies as defined above and/or a plurality of PPSFs as defined above.
- a pharmaceutical composition comprising a therapeutically effective amount of an antibody, or a fragment or derivative of an antibody as defined above and a pharmaceutically acceptable carrier.
- a method of treating or preventing Postpolio Syndrome comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody as defiend above.
- PPSFs Postpolio Syndrome features
- said determination of interaction between the candidate agent and the PPSF comprises quantitatively detecting binding of the candidate agent and the PPSF.
- a method of screening for or identifying agents that modulate the expression or activity of one or more more Postpolio Syndrome features comprising: (a) contacting a first population of cells expressing the PPSF with a candidate agent;
- PPSFs Postpolio Syndrome features
- said administration of a candidate agent results in an increase in the level of said PPSF(s), or mRNA encoding said PPSF(s), or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
- said administration of a candidate agent results in a decrease in the level of said PPSF(S), or mRNA encoding said PPSF(s), or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
- PPSFs Postpolio Syndrome features
- said PPSF(s) is a recombinant protein.
- said PPSF(s) is immobilized on a solid phase.
- a method for screening or diagnosis of Postpolio Syndrome in a subject or for monitoring the effect of an anti-Postpolio Syndrome drug or therapy administered to a subject comprising:
- oligonucleotide probe comprising 10 or more consecutive nucleotides complementary to a nucleotide sequence encoding an PPSF as defined above with RNA obtained from a biological sample from the subject or with cDNA copied from the RNA wherein said contacting occurs under conditions that permit hybridization of the probe to the nucleotide sequence if present;
- step (c) comparing the hybridization, if any, detected in step (b) with the hybridization detected in a control sample, or with a previously determined reference range.
- said step (a) includes the step of hybridizing the nucleotide sequence to a DNA array, wherein one or more members of the array are the probes complementary to a plurality of nucleotide sequences encoding distinct PPSFs.
- a method of treating or preventing Postpolio Syndrome comprising administering to a subject in need of such treatment or prevention a therapeutically effetive dose of an agent that modulates the activity of one or more of more Postpolio Syndrome features (PPSFs) as defined above; whereby the symptoms of Postpolio Syndrome are ameliorated.
- PPSFs Postpolio Syndrome features
- Example 1 PPS patients consisted of nine male patients with a mean age of 59,3 years (range 45-69), and six females with a mean age of 60,5 years (range 42-72).
- the OND group consisted of a variety of ages and symptoms.
- the mean age of the male OND patients were 42,1 years (range 19-74) and 38,6 years for the females (range 14-73).
- the PPS patients were carefully selected and had a definite history of acute poliomyelitis and new problems such as increased muscle weakness, muscle fatigue and pain in muscle groups earlier affected by poliomyelitis, according to the criteria for PPS as given by Halstead LS, and Rossi CD (Post-polio syndrome: clinical experience with 132 consecutive outpatients.
- the peak isometric (60°) muscular strength was measured in the quadriceps on the weakest side. The range was wide due to the fact that some persons had strength loss below the knee level. One patient had almost normal strength and walking capacity but fulfilled all PPS criteria and was in an early PPS stage.
- CSF samples were acquired with lumbar punction and thereafter the tubes were centrifuged and CSF without cells was frozen until further analysis. All CSF samples were affinity purified with POROS anti-HSA column, 2 ml (Applied Biosystems, USA) and a HiTrap Protein G column, 1 ml (GE healthcare, USA) to remove Albumin and IgG respectively. The samples were purified according to manufacturer's instructions and all solutions included Complete protease inhibitor cocktail (Roche, Germany). Each eluted sample was desalted and concentrated using Amnicon-Ultra spin column with a cut-off at 5 KDa (Millipore, USA).
- Detected protein spots were then matched between gels and a synthetic master image was prepared to represent a majority of the protein spots present in all gels and groups of samples.
- the quantity of each protein spot was expressed as ppm (parts per million) of the total sum of the integrated spot volumes of the given gel image.
- the filtering of spots was done with respect to the MS group: for dataset 1 the criterion was set that at least 40 % of the samples in either MS or control should be matched for a spot to be included in the dataset, this resulted in a dataset of 911 spots. For dataset 2, a somewhat stricter criterion was used: a spot should be matched in at least 50% of the samples in either MS or the control group. This resulted in a dataset of 787 spots.
- PLS Partial Least Squares
- VIP is a weighted sum of squares of the PLS weights, with the weights calculated from the amount of Y-variance of each PLS component in the model.
- the predictor matrix is the 2DE data
- the response is a binary vector denoting class membership, in this case zeros represents the OND/HC class and ones the PPS class.
- a cutoff needs to be set for the y, such that if the predicted y value is below the cutoff, the predicted class membership will be OND/HC in this case, and if the predicted y value is above the cutoff, the predicted class membership will be PPS.
- the PLS modeling was done in Matlab (The Mathworks, Inc) using the PLS toolbox (Eigenvector Research) Hierarchical clustering was performed in Spotfire in order to provide a visualisation of the data.
- Protein identification In-gel digestion Protein spots of interest were excised from gels using the EXQUEST spot cutter robot (Bio-Rad, Hercules, CA, USA), transferred to 96-well plates. Up to 6 protein spots of same spot number were pooled to each well in order to facilitate the identification of low abundant protein spots.
- the excised gel plugs were subjected to distaining using a wash solution consisting of 70% ACN in 25 mM ammonium bicarbonate. The gel plugs were incubated this wash solution for 10 minutes. This washing procedure was repeated three times. Finally, the wash solution was removed followed by speed vaccing for 20 minutes.
- High Sensitivity Micro-Tech Workstation for protein identification A micro technology workstation was used for high sensitivity protein analysis and identification of targets and biomarkers within the study. This microtech platform builds on chip integrated solid-phase microextraction array and a microdispenser for sample purification and trace enrichment of peptides as previously described (Wallman et al, Electrophoresis, 2004, 25, 3778-3787).
- the capillary mircofiuidic system is operated in an automated set-up.
- Chip- integrated sample clean-up of the protein sample performed in a 96-array chip format.
- the microextraction array was loaded with solid-phase media (Poros R2 50 ⁇ m beads) for purification and enrichment of proteomic samples.
- Samples bound to the microchip were eluted in a volume of 100 nL.
- the protein sample is eluted by utilizing a sequential capillary action that is docking to piezoelectric microdispencer.
- the subsequent transfer is made to the MALSI TOF mass spectrometry target plate where typically a burst of 1000 droplets of the sample is spotted onto the MALDI plate.
- the HPLC effluent was directly fractionated onto a 144 position ABI MALDI target plate using an Agilent micro fraction collector and spots were deposited every 30 seconds during the gradient (90 spots/run). The spots were allowed to dry completely prior to addition of I ⁇ L of CHCA matrix.
- the MS/MS data from the MALDI-TOF/TOF instrument was acquired accordinging to a method previously described by Zhen et al. (J. Am. Soc. Mass Spectrom. 2004, 15, 803-822).
- a standard peptide mixture containing 6 peptides diluted to about 500 fmol/ ⁇ L were applied on the six calibration spot positions of the target plate and used as external calibration points.
- MS/MS data obtained from the MALDI- TOF/TOF instrument were searched using Mascot as the search engine. All searches were performed against the human, rat, and mouse subset of in-house protein sequence databases (Genseq P, RefSeqP, PDB, PIR, SwissProt and TREMBL).
- the Global Proteome Server (GPS) was used for submitting data acquired from the TOF/TOF for database searching.
- the Mascot searching was performed using the default settings for the TOF/TOF instrument as supplied by Matrix Science (peptide mass tolerance of 50 ppm and a fragment mass tolerance of 0.2 Da). Oxidation (M) and carbamidomethyl were allowed as variable modifications.
- Results In total, 15 PPS samples and 43 OND/HC samples were included in the study. The patient samples originate from a larger study where the major focus was unbiased identification of biomarkers in Multiple sclerosis. PPS patient samples were included to serve as reference disease material together with controls OND/HC. We analysed in total 1499 2D-gel separated protein spots within the large study and 5-13 protein spots with very high predictive power for the PPS group were observed, that we then tested on a separate set of OND and PPS samples The predictive modelling was carried out in two steps. First, the dataset of 1499 spots that contained 36 OND/HC and 9 PPS samples was used to select the spots with the highest predictivity and optimise model parameters. This set of samples will be referred to as the model set.
- the model optimisation and spot selection was done through a crossvalidation/bootstrapping procedure described more in detail below.
- the predictivity of the model and the associated selected 13 spots was evaluated using a new set of samples, a test set, consisting of 7 OND and 6 PPS samples. See Figure 1 for an illustration of the datasets and how they were used in the analysis.
- the gels for the samples used to build the model were subjected to a second round of matching for the selected 13 spots, and missing values can now be considered to arise from that they are below the detection limit.
- VIP variable importance weights
- the model optimization was performed on two datasets, resulting from two different approaches to handling missing values (see materials & methods).
- missing values were considered to arise from that the protein levels were below the detection limit, and were replaced by a low value.
- missing values were replaced using the LS impute algorithm
- the PLS models with one component gave the best results for both datasets, and the prediction rate remained relatively constant as variables were dropped and it was seen that about 15-5 variables was sufficient to achieve a good prediction rate.
- Variable ranks from the two models were combined to form an overal variable importance rank, and the top 13 variables were chosen for the final model.
- the corresponding protein spots were cut out from 2D-gels and identified by masspectrometry (Table 1) as described above.
- a ROC analysis was performed to find the cutoff that gave a balanced prediction rate, i.e. the proportion correctly classified samples is equal between the two classes. This resulted in a y cutoff at 0.35 for the model using the background- value replacement for missing values and 0.5 for the dataset that was imputed using LS Impute.
- the average prediction rate in the crossvalidation/bootstrap procedure was 90% and 99% correctly predicted samples for the two datasets, respectively.
- a representative 2D-gel is shown in figure 3 and the locations of top- 13 protein spots are zoomed in. Each spot is given a unique database SSP number by the PDQuest software. Table 1 show protein identities obtained by mass spectrometry analysis and estimated molecular weight/isoelectric point.
- a PLS model was built on the set of samples that were in the first set of analysis.
- the training set contained 9 PPS samples and 36 OND/HC samples and the test set contained 6 new PPS samples and 7 new OND samples.
- the parameters were selected as described above: 1 PLS component and the cutoff was chosen to be between 0.35 and 0.5 (based on the cutoffs obtained in the model optimization).
- the dataset Prior to building the model, the dataset had gone through a second matching round for the selected 13 spots, and missing values can now be considered to arise from that they are below the detection limit.
- Table 2 shows the VIP, VIP rank and regression coefficients for the complete and the training datasets.
- the values based on the complete dataset are similar to those for the is training dataset, however the rank for some of the spots in the middle range has been changed. This indicates the differences in VIP between these spots are minor, and that too much confidence cannot be placed in a specific rank number. However, the two top and the two bottom ranks are the same between the two datasets.
- the top 11 -ranked protein based on VIP values are represented by only three proteins; gelsolin, kallikrein 6 and hemopexin. It should be stressed that one need all proteins together to obtain maximal prediction. PAM and GSHB scores the lowest values and it is likely these are of minor importance for the model.
- a type of sensitivity analysis was carried out in which variables were dropped successively one variable at a time (based on VIP rank, Table 3), models built on the training dataset and recording the prediction rate for the test set.
- the prediction rate as a function of the number of variables (at cutoff 0.425) showed to be stable down to using just one variable for the OND/HC class, and down to 5 variables for the PPS class. At 4-1 variables the PPS class prediction drops from 6/6 to 5/6. Thus, the prediction is stable in terms of retaining the predictive ability when variables are dropped from the models.
- Gelsolin is represented by fragments only, indicating that the quantity of full-length variant of gelsolin do not add value in a predictive model.
- Hemopexin show on the other hand a clear decrease of the full-length variant in parallel with an increase of fragmented hemopexin.
- the kallikrein 6 protein spot is likely to be a full-length variant (according to observed versus theoretical molecular weight).
- Hierarchical clustering is an unsupervised method, which allows data to selforganise according to similarity. A clear separation of the two patient groups was observed, and all samples except one (# 23) cluster within the correct diagnostic group. This is the same sample that was incorrectly classified in the PLS model. Interestingly, sample #23 originate from the only patient diagnosed with fibromyalgia, which indicate that the top- 13 identified protein described above may be used as biomarkers for this disease.
- a method for screening or diagnosis of fibromyalgia in a subject for determining the stage or severity of fibromyalgia in a subject, for identifying a subject at risk of developing fibromyalgia, or for monitoring the effect of therapy administered to a subject having fibromyalgia, said method comprising:
- said array comprises one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
- PPS Features PPS Features
- a method for screening or diagnosis of fibromyalgia in a subject for determining the stage or severity of fibromyalgia in a subject, for identifying a subject at risk of developing fibromyalgia, or for monitoring the effect of therapy administered to a subject having fibromyalgia, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following proteins: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB.
- said method comprises quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
- PPS Features PPS Features
- said body fluid is cerebrospinal fluid (CSF).
- CSF cerebrospinal fluid
- said body fluid is plasma
- said body fluid is serum. In another embodiment of this aspect, said body fluid is urine.
- Fig 3a Box-plots showing expression levels in ppm for 43 OND/HC and 15 PPS samples. The boxes are defined by the 25th and 75th percentile, thus 50 percent of the data is represented within each box. Upper and lower adjacent values, are represented by whiskers (defined by SpotfireTM).
- Table 3 a and b show mean, standard deviations, coefficent of variation (CV%) within the OND/HC and PPS groups, as well as the fold change, calculated as the ratio of the mean levels.
- Table 3a Mean, standard deviations, coefficent of variation (CV%) within the OND/HC and PPS groups, as well as the fold change, calculated as the ratio of the mean levels.
- Gelsolin- fragments are the most prominent proteins present in the CSF of persons with PPS. Gelsolin is tightly associated with apoptosis, and both pro-apoptotic and anti- apoptotic roles have been described. As to the pro-apoptotic pathaway gelsolin interacts with the caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases which in turn has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3.
- CPP32 caspase-3
- YAMA cysteinyl proteases
- Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severedactin filaments in vitro in a Ca 2+ -independent manner. Expression of the gelsolincleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.
- Hemopexin is a 80-Kda glycoprotein mainly expressed in the liver and it belongs to the family of acute-phase proteins induced during inflammatory events (Melchers W, de Visser M, Jongen P, et al. The postpolio syndrome: no evidence for poliovirus persistence. Ann Neurol 1992;32(6):728-32]).
- the most well characterized function of hemopexin is to bind heme and to protect against oxidative stress by receptor-mediated induction of heme oxygenase (Jubelt B, Salazar-Grueso EF, Roos RP, Cashman NR. Antibody titer to the poliovirus in blood and cerebrospinal fluid of patients with post-polio syndrome.
- Kallikrein 6 was the third most prominently altered protein in the CSF of persons with PPS. There was an approximate threefold increase of its levels in PPS as compared to both OND and SPMS. In addition to this single kallikrein 6 spot (SSP 8221) we identified one other spot (SSP8209) as kallikrein 6 (data not shown), although at lower prediction scores (VIP rank #35). However, both kallikrein 6 spots showed increased levels in PPS, which support the possible impact of kallikrein 6 in PPS. Kallikrein 6 is a serine protease belonging to a gene family with 15 members and is preferentially expressed in the CNS and at highest levels in the spinal cord and brain stem (Diamandis, E.
- Kallikrein 6 is expressed in neurons, oligodendrocytes, and strongly up-regulated in astrocytes, macrophages/microglia in CNS pathologies such as spinal cord trauma and in human or experimental inflammatory demyelinating disease. Hence, its up-regulation can be regarded as a part of a CNS innate immunity response to a variety of insults, both traumatic and primary inflammatory events. It has also been detected in the CSF/CNS of neurodegenerative diseases such as Alzheimers and
- Parkinsons disease (Diamandis EP, Yousef GM, Petraki C, Soosaipillai AR. Human kallikrein 6 as a biomarker of alzheimer's disease. Clin Biochem. 2000 Nov;33(8):663-7. PMID: 11166014).
- kallikrein 6 was detected as a full-length protein and is accessible for measurements with ELISA it constitutes an attractive candidate as biomarker in PPS to be validated in larger materials.
- hK6 human kallikrein 6
- the invention described in detail provides methods and compositions for clinical screening and diagnosis of Postpolio Syndrome (PPS), in a mammalian subject for identifying patients most likely to respond to a particular therapeutic treatment, for monitoring the results of PPS therapy, for drug screening and drug development.
- PPS Postpolio Syndrome
- the invention also encompasses the administration of therapeutic compositions to a mammalian subject to treat or prevent PPS.
- the mammalian subject may be a non-human mammal, but is preferably human.
- the invention will be described with respect to the analysis of CSF samples.
- a body fluid for example but without limitation: blood, serum, plasma, saliva or urine
- tissue sample from a subject at risk of having or developing PPS or homogenate thereof.
- the methods and compositions of the present invention are useful for screening and diagnosis of a living subject, but may also be used for postmortem diagnosis in a subject, for example, to identify family members of the subject who are at risk of developing the same disease.
- Feature refers to a spot identified in a 2D gel
- Postpolio Syndrome feature PPSF
- PPSF Postpolio Syndrome feature
- a feature or spot identified in a 2D gel is characterized by its isoelectric point (pi) and apparent molecular weight (MW) as determined by 2D gel electrophoresis.
- a feature is "differentially expressed" in a first sample or sample set with respect to a second sample or sample set when a method for detecting the said feature (e.g., 2D electrophoresis) gives a different signal when applied to the first and second samples or sample sets.
- a method for detecting the said feature e.g., 2D electrophoresis
- the relative abundance of a feature in the two samples or sample sets is determined in reference to its normalized signal, in two steps.
- the signal obtained upon detecting the feature in a first sample or sample set is normalized by reference to a suitable background parameter, e.g., (a) to the total protein in the sample being analyzed (e.g., total protein loaded onto a gel); (b) to an Expression Reference Feature (ERF) i.e., a feature whose abundance is substantially invariant, in the population of subjects being examined.
- a suitable background parameter e.g., (a) to the total protein in the sample being analyzed (e.g., total protein loaded onto a gel); (b) to an Expression Reference Feature (ERF) i.e., a feature whose abundance is substantially invariant, in the population of subjects being examined.
- ERP Expression Reference Feature
- the normalized signal for the feature in the first sample or sample set is compared with the normalized signal for the same feature in the second sample or sample set in order to identify features that are "differentially expressed" in the first sample or sample set with respect to the second sample or sample set.
- Variant refers to a polypeptide which is a member of a family of polypeptides that are encoded by a single gene or from a gene sequence within a family of related genes and which differ in their pi or MW, or both. Such variants can differ in their amino acid composition (e. g. as a result of alternative mRNA or premRNA processing, e.g. alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from 10 differential post-translational modification (e.g., glycosylation, acylation, phosphorylation) .
- differential post-translational modification e.g., glycosylation, acylation, phosphorylation
- PPSF analog refers to a polypeptide that possesses similar or identical function(s) as a PPSF but need not necessarily comprise an amino acid sequence that is similar or identical to the amino acid sequence of the PPSF, or possess a structure that is similar or identical to that of the PPSF.
- an amino acid sequence of a polypeptide is "similar" to that of a PPSF if it satisfies at least one of the following criteria: (a) the polypeptide has an amino acid sequence that is at least 30 To (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of the PPSF; (b) the polypeptide is encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding at least 5 amino acid residues (more preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues
- a polypeptide with "similar structure" to that of 35 a PPSF refers to a polypeptide that has a similar secondary, tertiary or Quarternary structure as that of the PPSF.
- the structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, mass spectrometry and crystallographic electron microscopy.
- Chimeric Antibody refers to a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a marine mAb. (See, e.g., Cabilly et al., U.S. Patent No. 10 4,816,567; and Boss et al., U.S. Patent No. 4,816397, which are incorporated herein by reference in their entirety.)).
- a portion of the antibody may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHl, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric antibodies.
- Humanised Antibody refers to a molecule from non-human species having one or 15 more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
- “Derivative” refers to a polypeptide that comprises an amino acid sequence of a second polypeptide that has been altered by the introduction of at least one amino acid residue substitution, deletion or addition.
- the derivative polypeptide possesses a similar or identical function as the second polypeptide.
- “Fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid 25 residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a second polypeptide.
- the fragment of a PPSF possesses the functional activity of the PPSF.
- the "percent identity" of two amino acid sequences or of two nucleic acid sequences can be or is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in either sequences for best alignment with the other sequence) and comparing the amino acid residues or nucleotides at corresponding positions.
- the "best alignment” is an alignment of two sequences that results in the highest percent identity.
- the determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
- An example of a 40 mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
- the NBLAST and XBLAST programs of Altschul, et al. (1990) J. MoI. Biol. 215:403-410 have incorporated such an algorithm.
- Gapped BLAST can be 5 utilized as described in Altschul et al. (1997) Nucleic Acids Res.
- PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
- BLAST Altschul et al.
- Gapped BLAST e.g., XBLAST and NBLAST
- the default parameters of the respective programs e.g., XBLAST and NBLAST
- Diagnosis refers to diagnosis, prognosis, monitoring, characterizing, selecting patients, including participants in clinical trials, and identifying patients at risk for or having a particular disorder or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment.
- Treatment refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure the infirmity or malady in the instance where the patient is afflicted.
- Agent refers to all materials that may be used to prepare pharmaceutical and diagnostic compositions, or that may be compounds, agonists, antagonists, nucleic acids, polypeptides, fragments, isoforms, variants, or other materials that may be used independently for such purposes, all in accordance with the present invention.
- CSF Cerebrospinal fluid
- “Serum” refers to the supernatant fluid produced by clotting and centrifugal 40 sedimentation of a blood sample.
- Pulsma refers to the supernatant fluid produced by inhibition of clotting (for example, by citrate or EDTA) and centrifugal sedimentation of a blood sample.
- Blood as used herein includes serum and plasma.
- Two-dimensional electrophoresis (2D -electrophoresis) means a technique comprising isoelectric focusing, followed by denaturing electrophoresis; this generates a two- dimensional gel (2D-gel) containing a plurality of separated proteins.
- two-dimensional electrophoresis is used to analyze CSF from a subject, preferably a living subject, in order to detect or quantify the expression of one or more Postpolio Syndrome Features (PPSFs), screening, prevention or diagnosis of PPS, to determine the prognosis of a subject having PPS, to monitor progression of PPS, to monitor the effectiveness of PPS therapy, for identifying patients most likely to respond to a particular therapeutic treatment, or for drug development.
- PPSFs Postpolio Syndrome Features
- a number of samples from subjects having PPS and samples from subjects free from PPS are separated by two-dimensional electrophoresis, and the fluorescent digital images of the resulting gels are matched to a chosen representative primary master gel image.
- This process allows any gel feature, characterized by its pi and MW, to be identified and examined on any gel of the study.
- the amount of protein present in a given feature can be measured in each gel; this feature abundance can be averaged amongst gels from similar samples (e.g. gels from samples from subjects having PPS).
- statistical analyses can be conducted on the thus created sample sets, in order to compare 2 or more sample sets to each other.
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Abstract
The present invention relates to methods for screening or diagnosis of Postpolio Syndrome (PPS) in subjects, for determining the stage or severity of PPS, for identifying subjects at risk of developing PPS, or for monitoring the effect of therapy administered to a subject having PPS. The application also discloses methods for screening or diagnosis of fibromyalgia.
Description
A METHOD FOR SCREENING OR DIAGNOSIS OF POSTPOLIO SYNDROME AND FIBROMYALGIA
The present invention relates to the identification of polypeptides, proteins and protein isoforms that are associated with Postpolio Syndrome (PPS), and to their use for e.g., clinical screening, diagnosis, prognosis, therapy and prophylaxis, as well as for drug screening and drug development.
The present invention provides methods and compositions for clinical screening, diagnosis and treatment of PPS, for monitoring the effectiveness of PPS treatment, for selecting participants in clinical trials, for identifying patients most likely to respond to a particular therapeutic treatment and for screening and development of drugs for treatment of PPS. The Post-Polio Syndrome (PPS) is a condition appearing several decades after the acute poliomyelitis infection. Survivors of acute paralytic disease often develop new or increased muscle weakness, fatigue and pain from the musculoskeletal system (Halstead LS, Rossi CD, Orthopedics, 1985: 845-50). Even other symptoms as breathing difficulties, swallowing problems and other brain stem symptoms may appear. An ongoing denervation, may be the most important reason for the increased weakness in muscles earlier affected by poliomyelitis (McComas AJ, et al. Brain 1997;120 ( Pt 8):1415-21; Grimby G. et al. Muscle Nerve 1998;21(11): 1428-37). The denervation is highly likely to depend on an ongoing loss of motorneurons, which is faster and begins at an earlier age compared to an average population (Grimby et al, supra). Denervation is compensated by reinnervation by means of collateral sprouting. This compensatory mechanism leads to an increase of motor unit size (Grimby et al. supra). At the end reinnervation cannot compensate for denervation, leading to insufficient compensation, and decrease of muscle strength follows.
The causes of the ongoing loss of motor neurons in PPS are, however, unclear, but we hypothesize that chronic intrathecal inflammation is one potential cause. It has been shown that patients with PPS exhibit an increased expression of mRNA for proinflammatory cytokines in cerebrospinal fluid (CSF) (Gonzalez H, et al. J Neurol Sci 2002; 205(l):9-13;
Gonzalez H, et al. J Neuroimmunol 2004; 150(1-2): 139-44) pointing to an inflammatory process in the central nervous system (CNS). A further support for an inflammatory process is perivascular and lymphocyte infiltrates in the parenchyma and active gliosis in the spinal cord (Miller DC. Ann N Y Acad Sci 1995;753:186-9; Dalakas MC, et al. N Engl J Med 1987;317(25): 1602-3) as well as signs of inflammation in affected muscles (Dalakas M and Ilia I Adv Neurol 1991;56:495-511; Dalakas M, Neurology 1988;38(l):99-104). It is known from animal experiments that overproduction or aberrant cytokine production may cause nervous tissue damage (Sun D, et al. Neuropathol Appl Neurobiol 2004;30(4):374-84). Data from recent studies suggests that IVIG treatment is able to halt the inflammatory process and have beneficial clinical effects in PPS (Gonzalez H, et al. J Neurol Sci 2002; 205(l):9-13), indirectly supporting the thesis of an inflammatory process causing motor neuron death in PPS. However, the reason for the chronic intrathecal inflammation in PPS remains unclear. The following options are considered: 1) a continuous immune response against persistent, perhaps mutated poliovirus, 2) a defective down-regulation of the original antiviral response, or 3) an autoimmune, or otherwise aberrant response induced by the virus or by the neurodegeneration directed against unknown neuronal, or non-neuronal autoantigens. To learn more about the pathogenesis of PPS, an unbiased technique was applied in form of detection of a braod spectrum of proteins in the CSF from persons with PPS, likely to reflect events in the tissue related to inflammation and/or neurodegeneration. As comparison 43 patients with other non-inflammatory disorders (OND) were evaluated.
Survivors of poliomyelitis often develop increased or new symptoms decades after the acute infection, known as the post-polio syndrome (PPS). Patients display fatigue, pain and decrease of muscle strength, based in an ongoing denervation/neurodegeneration. The cause(s) of these late sequele are unknown, but a role of proinflammatory cytokines have been suggested since they are produced at increased levels within the central nervous system. The inventors have studied the proteomic profile in cerebrospinal fluid (CSF) in persons with PPS, compared to control subjects to obtain further information on the pathogenic mechanisms in this condition.
15 patients with well-defined Postpolio Syndrome (PPS) were analysed for 1499 proteins in their cerebrospinal fluid and were compared to 43 patients with other non-inflammatory disorders (OND). The OND group consisted of patients with pain syndromes, vertigo, sensory symptoms, and fatigue. One patient suffered from psychosis. Ten healthy controls were included. Thus, the CSF of persons with PPS was characterized by differential expression of 5 distinct proteins: gelsolin, hemopexin, peptidylglycine alpha-hydroxylating monooxygenase (PAM), glutathione synthetase (GSHB), and kallekrein 6. These proteins are involved in apoptosis and/or inflammation. The data provide strong evidence that the clinical deteroriation during PPS is due to an active inflammatory neurodegeneration, of potential therapeutic relevance. The data set the stage for diagnostic and/or prognostic evaluation of these proteins.
DESCRIPTION OF THE DRAWINGS
Figure 1. Data analysis strategy. The model optimisation was performed on a set of 1499 spots that included 36 OND/HC and 9 PPS samples. In the optimization procedure, this set of samples was randomly divided into training and test sets, repeatedly, to obtain an estimate of the prediction performance. From this analysis, the spots were ranked by importance in the model, and the top 13 spots that were identified using mass spectrometry, were chosen to constitute the final model. The samples used to train the model, were the 36+9 samples used in the model optimization, and this model was tested on a new independent set of samples, 6 PPS and 7 OND samples.
Figure 2. 2D-gel separation of CSF proteins from one PPS patient. The positions of the top- 13 identified protein spots are shown, hemopexin, gelsolin, kallikrein-6, peptidylglycine alpha-hydroxylating monooxygenase (PAM) and glutathione synthetase (GSHB). For reference, the positions of sero transferrin, alpha- 1 -antitrypsin and apo- lipoprotein Al are indicated. The right panel show zoom-in areas of identified proteins and SSP number. Mass spectrometry data for the top- 13 proteins are shown in Table 1.
Figure 3. Box-plots showing expression levels in ppm for 43 OND/HC and 15 samples. The boxes are defined by the 25th and 75th percentile, thus 50 percent of the data is represented within each box. Upper and lower adjacent values, are represented by whiskers
(defined by Spotfire™). The median is represented by blue arrows, the mean is represented by black arrows.
The present invention originates from a study that originates from observations in another study. All patient samples were first analysed in a study focusing on unbiased identification of biomarkers in multiple sclerosis (manuscript in preparation). In that study, PPS patient samples were included to serve as a reference disease material. However, results from the PPS group were very interesting and deserves to be presented separately and with the aims given above. We analysed 1499 2D-gel separated protein spots within the large study and 5-13 protein spots with very high predictive power for the PPS group was observed. The predictivity of the top 13 spots, that also were identified using mass spectrometry, were evaluated on an independent test set (Fig 1).
There has now been identified a number of proteins and /or fragments thereof, that are distinctively indicative for Postpolio Syndrome. Therefore, in one aspect of the invention, there is provided a method for screening or diagnosis of Postpolio Syndrome (PPS) in a subject, for determining the stage or severity of PPS in a subject, for identifying a subject at risk of developing PPS, or for monitoring the effect of therapy administered to a subject having PPS, said method comprising:
(a) analysing a test sample of body fluid from the subject by two-dimensional electrophoresis to generate a two-dimensional array of features, said array comprising one or more of the following protiens: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB.
(b) comparing the abundance of the one or more PPSFs in the test sample with the abundance of the one or more of said proteins in a body fluid sample from one or more subjects free from PPS, or with a previously determined reference range for that feature in subjects free from PPS, or with the abundance at least one Expression Reference Feature (ERF) in the test sample.
In one embodiment of this aspect, said array comprises one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
In another aspect of the invention, there is provided a method for screening or diagnosis of Postpolio Syndrome (PPS) in a subject, for determining the stage or severity of PPS in a subject, for identifying a subject at risk of developing PPS, or for monitoring the effect of therapy administered to a subject having PPS, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following proteins: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB.
In one embodiment of this aspect, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
In another aspect of the invention, said body fluid is cerebrospinal fluid (CSF).
In another aspect of the invention, said body fluid is plasma.
In another aspect of the invention, said body fluid is serum.
In another aspect of the invention, said body fluid is urine.
In another aspect of the invention, there is provided an antibody capable of immunospecific binding to a Postpolio Syndrome Feature (PPSF).
In one embodiment of this aspect, said antibody is a monoclonal antibody.
In another embodiment of this aspect, said antibody is a chimeric antibody.
In another embodiment of this aspect, said antibody is a bispecific antibody.
In another embodiment of this aspect, said antibody is a humanised antibody.
In another aspect of the invention, there is provided a kit comprising one or more antibodies as defined above and/or one or more PPSF as defined above, other reagents and instructions for use.
In one embodiment of this aspect, said kit is for use in the screening or diagnosis of Postpolio Syndrome in a subject, for determining the stage or severity of Postpolio
Syndrome in a subject, for identifying a subject at risk of developing Postpolio Syndrome, or for monitoring the effect of therapy administered to a subject having Postpolio Syndrome.
In one embodiment of this aspect, said kit comprising a plurality of antibodies as defined above and/or a plurality of PPSFs as defined above.
In another aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an antibody, or a fragment or derivative of an antibody as defined above and a pharmaceutically acceptable carrier.
In another aspect of the invention, there is provided a method of treating or preventing Postpolio Syndrome comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody as defiend above.
In another aspect of the invention, there is provided a method of screening for agents that interact with one or more Postpolio Syndrome features (PPSFs) as defined above, said method comprising:
(a) contacting an PPSF with a candidate agent; and
(b) determining whether or not the candidate agent interacts with the PPSF.
In one embodiment of this aspect, said determination of interaction between the candidate agent and the PPSF comprises quantitatively detecting binding of the candidate agent and the PPSF.
In another aspect of the invention, there is provided a method of screening for or identifying agents that modulate the expression or activity of one or more more Postpolio Syndrome features (PPSFs), comprising:
(a) contacting a first population of cells expressing the PPSF with a candidate agent;
(b) contacting a second population of cells expressing said PPSF with a control agent; and
(c) comparing the level of said PPSF(s) in the first and second populations of cells, or comparing the level of induction of a downstream effecter in the first and second populations of cells.
In another aspect of the invention, there is provided a method of screening for or identifying agents that modulate the expression or activity of one or more more Postpolio Syndrome features (PPSFs) as defined above, said method comprising:
(a) administering a candidate agent to a first mammal or group of mammals;
(b) administering a control agent to a second mammal or group of mammals; and
(c) comparing the level of expression of the PPSF, or mRNA encoding said PPSF in the first and second groups, or comparing the level of induction of a downstream effecter in the first and second groups.
In one embodiment of this aspect, said administration of a candidate agent results in an increase in the level of said PPSF(s), or mRNA encoding said PPSF(s), or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
In another embodiment of this aspect, said administration of a candidate agent results in a decrease in the level of said PPSF(S), or mRNA encoding said PPSF(s), or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
In another aspect of the invention, there is provided a method of screening for or identifying agents that modulate the activity of one or more of the more Postpolio Syndrome features (PPSFs) as defined above, said method comprising:
(a) in a first aliquot, contacting a candidate agent with the PPSF(s) and;
(b) determining and comparing the activity of the PPSF(s) in the first aliquot after addition of the candidate agent with the activity of PPSF(s) in a control aliquot, or with a previously determined reference range.
In another embodiment of this aspect, said PPSF(s) is a recombinant protein.
In another embodiment of this aspect, said PPSF(s) is immobilized on a solid phase.
In another aspect of the invention, there is provided a method for screening or diagnosis of Postpolio Syndrome in a subject or for monitoring the effect of an anti-Postpolio Syndrome drug or therapy administered to a subject, comprising:
(a) contacting at least one oligonucleotide probe comprising 10 or more consecutive nucleotides complementary to a nucleotide sequence encoding an PPSF as defined above with RNA obtained from a biological sample from the subject or with cDNA copied from the RNA wherein said contacting occurs under conditions that permit hybridization of the probe to the nucleotide sequence if present;
(b) detecting hybridization, if any, between the probe and the nucleotide sequence; and
(c) comparing the hybridization, if any, detected in step (b) with the hybridization detected in a control sample, or with a previously determined reference range.
In one embodiment of this aspect, said step (a) includes the step of hybridizing the nucleotide sequence to a DNA array, wherein one or more members of the array are the probes complementary to a plurality of nucleotide sequences encoding distinct PPSFs.
In another aspect of the invention, there is provided a method of treating or preventing Postpolio Syndrome comprising administering to a subject in need of such treatment or prevention a therapeutically effetive dose of an agent that modulates the activity of one or more of more Postpolio Syndrome features (PPSFs) as defined above; whereby the symptoms of Postpolio Syndrome are ameliorated.
The present invention will now be illustrated with the following non-limiting examples:
Example 1 PPS patients consisted of nine male patients with a mean age of 59,3 years (range 45-69), and six females with a mean age of 60,5 years (range 42-72). The OND group consisted of a variety of ages and symptoms. The mean age of the male OND patients were 42,1 years (range 19-74) and 38,6 years for the females (range 14-73). The PPS patients were carefully selected and had a definite history of acute poliomyelitis and new problems such as increased muscle weakness, muscle fatigue and pain in muscle groups earlier affected by poliomyelitis, according to the criteria for PPS as given by Halstead LS, and Rossi CD (Post-polio syndrome: clinical experience with 132 consecutive outpatients. Birth Defects Orig Artie Ser 1987;23(4): 13-26), age between 18 and 75. Further criteria were added as BMI <30 and a stable weight, as obesity or weight differences per se could have impact on disability. Other diseases as hypothyreosis, diabetes (not fully controlled) and every other medical or ortophedic disorder that could give rise to symptoms mimicking PPS were excluded. All PPS-patients were ambulatory. The mean time between the acute polio infection and the present examination was 43 years (range 34-51 years). The mean time since the diagnosis of PPS was 6.5 years (range 1-14 years).
The peak isometric (60°) muscular strength was measured in the quadriceps on the weakest side. The range was wide due to the fact that some persons had strength loss below the knee level. One patient had almost normal strength and walking capacity but fulfilled all PPS criteria and was in an early PPS stage.
All 15 PPS patients showed elevated mRNA expression of TNF-α in CSF.
All patients had loss of motor units and motor units with increased amplitude and duration on the electrophysiological examination, i.e. compatible with prior poliomyelitis.
The OND patients (n=43) were recruited from the outpatient clinic at The Karolinska
Hospital. These patients were regarded as non-inflammatory and the lumbar puncture was performed as a part of the routine clinical analysis of these patients. The CSFs of these
patients displayed no signs of inflammation in form of pleocytosis, invreased IgG index or oligoclonal bands.
The symptomology of these patients was very varied. In addition CSFs from a group (n=10) of healthy controls recruited as controls in context with another study of headache, were at hand for analysis.
A comparison with samples from persons with secondary progressive multiple sclerosis (SPMS) was added (n=17). SPMS samples were age matched to the PPS group and can be considered as an inflammatory disease reference material. SPMS samples were also recruited from the Karolinska Hospital outpatient clinic. The mean age of the male SPMS patients (n=6) was 54.1 years (range 45-65) and 56.5 years for the female (n=l 1) patients (range 39-82). All samples were part of a larger study described elsewhere (manuscript in preparation).
CSF samples were acquired with lumbar punction and thereafter the tubes were centrifuged and CSF without cells was frozen until further analysis. All CSF samples were affinity purified with POROS anti-HSA column, 2 ml (Applied Biosystems, USA) and a HiTrap Protein G column, 1 ml (GE healthcare, USA) to remove Albumin and IgG respectively. The samples were purified according to manufacturer's instructions and all solutions included Complete protease inhibitor cocktail (Roche, Germany). Each eluted sample was desalted and concentrated using Amnicon-Ultra spin column with a cut-off at 5 KDa (Millipore, USA). Protein concentration by was determined using the Bradford protein kit (Bio-Rad, Hercules, CA, USA) and image analysis (Quantity One, Bio-Rad, Hercules, CA, USA) of Sypro Ruby® stained (Molecular Probes, Inc. USA) one-dimensional SDS- PAGE.
Two-dimensional gel electrophoresis (2DE) and image analysis Samples were solubilized in rehydration solution (Urea 8 M (Sigma, USA) DTT 19.5 mM, NP-40 (10%) 0.5 % (v/v) (USB Corporation, USA) IPG-buffer 4-7, 0.5 % (v/v) (GE healthcare, USA) Glycerol 7 % (v/v) CHAPS 1.5 % (Genomic solutions, USA), thiourea
2M, (Fluka, Germany)) and analysed by 2DE as described elsewhere (Newcombe J. et al. Journal of Chromatography B: Analytical Technologies in the Biomedical & Life Sciences. 815(l-2):191-202, 2005 Feb 5). All samples were analyzed in a randomized sequence to avoid batch-to-batch influence of the results. Briefly, isoelectric focusing was performed using 24 cm IPG strips pH 4-7L (GE Healthcare, USA), and the second dimension separation was conducted using 1.0 x 220 x 200 mm 12,5%T SDS-PAGE. 2D-gels were stained with Sypro Ruby® (Schulenberg, B. et al. Electrophoresis, 25(15): 2526-32 (2004)), and finally scanned at 100 μm resolution (Molecular Imager FX, Bio-Rad, Hercules, CA, USA). Image files were processed using the software PDQuest (version 7.3, Bio-Rad, Hercules, CA, USA). Detected protein spots were then matched between gels and a synthetic master image was prepared to represent a majority of the protein spots present in all gels and groups of samples. The quantity of each protein spot was expressed as ppm (parts per million) of the total sum of the integrated spot volumes of the given gel image.
Data analysis
The image analysis resulted in 1499 detected and matched spots for the whole matchset, that included the MS samples as well as the 9 PPS and 36 OND/HC samples. Due to biological and experimental variation it is not possible to match all spots in all gels. For example, in some cases the spot can be missing due to low expression level and in other cases the spot is present on the gel, but has been missed out during the matching. Thus, there will be values that are missing for some spots in some gels. Missing values are represented as zeros in the data table, regardless of the reason the spot is missing. Therefore, in the data analysis, two approaches were taken to handling missing values that resulted in two datasets. In one dataset, all zeros were replaced by a low value (2 ppm) that could be considered as a background value (zeros were replaced to allow data to be log transformed) - dataset 1. In the other approach, data were imputed using a model-based approach, that utilises information on correlations between spots and/or gels in replacing missing values [LS impute: accurate estimation of missing values in microarray data with least squares methods Nucleic Acids Research 2004 VoI 32 No 3 Hellem Bø T., Dysvik B., Jonassen L] - dataset 2. Since the PPS were initially included only to serve as a reference
group for the MS analysis, the filtering of spots was done with respect to the MS group: for dataset 1 the criterion was set that at least 40 % of the samples in either MS or control should be matched for a spot to be included in the dataset, this resulted in a dataset of 911 spots. For dataset 2, a somewhat stricter criterion was used: a spot should be matched in at least 50% of the samples in either MS or the control group. This resulted in a dataset of 787 spots.
Data was log-transformed and batch correction was performed on a per spot basis using a mixed-models approach to estimate batch effects (Applied Mixed Models in Medicine, Brown, H. and Prescott, R. Wiley 2nd ed 2006) prior to multivariate modelling.
Partial Least Squares (PLS) was used to model the data. (PLS-regression: a basic tool of chemometrics Chemometrics and Intelligent laboratory systems (Wold S, Sjostrom M, Eriksson, L, 2001 58 109-130) modelling. It is a regression method that reduces the dimensions in the data through maximising the covariance of the predictor matrix (X) with the response matrix (Y), keeping the corresponding dimensions (PLS components/latent variables). It is possible to identify variables that are important for the relationship between X and Y. One such variable importance measure is the Variable Importance in the Projection (VIP) parameter. VIP is a weighted sum of squares of the PLS weights, with the weights calculated from the amount of Y-variance of each PLS component in the model. Here, the predictor matrix is the 2DE data, and the response is a binary vector denoting class membership, in this case zeros represents the OND/HC class and ones the PPS class. To determine the class memberships for new samples predicted with the model, a cutoff needs to be set for the y, such that if the predicted y value is below the cutoff, the predicted class membership will be OND/HC in this case, and if the predicted y value is above the cutoff, the predicted class membership will be PPS. The PLS modeling was done in Matlab (The Mathworks, Inc) using the PLS toolbox (Eigenvector Research) Hierarchical clustering was performed in Spotfire in order to provide a visualisation of the data.
Protein identification
In-gel digestion: Protein spots of interest were excised from gels using the EXQUEST spot cutter robot (Bio-Rad, Hercules, CA, USA), transferred to 96-well plates. Up to 6 protein spots of same spot number were pooled to each well in order to facilitate the identification of low abundant protein spots. The excised gel plugs were subjected to distaining using a wash solution consisting of 70% ACN in 25 mM ammonium bicarbonate. The gel plugs were incubated this wash solution for 10 minutes. This washing procedure was repeated three times. Finally, the wash solution was removed followed by speed vaccing for 20 minutes. About 15 μL trypsin solution (0,075 μg) was added to the gel plugs and digestion was performed over night at 37°C. Finally, extraction was performed during 1 hour by adding 20 μL of extraction solution (1 % ACN, 0.1% TFA).
High Sensitivity Micro-Tech Workstation for protein identification: A micro technology workstation was used for high sensitivity protein analysis and identification of targets and biomarkers within the study. This microtech platform builds on chip integrated solid-phase microextraction array and a microdispenser for sample purification and trace enrichment of peptides as previously described (Wallman et al, Electrophoresis, 2004, 25, 3778-3787).
Briefly, the capillary mircofiuidic system is operated in an automated set-up. Chip- integrated sample clean-up of the protein sample, performed in a 96-array chip format. The microextraction array was loaded with solid-phase media (Poros R2 50 μm beads) for purification and enrichment of proteomic samples. Samples bound to the microchip were eluted in a volume of 100 nL. Next, the protein sample is eluted by utilizing a sequential capillary action that is docking to piezoelectric microdispencer. The subsequent transfer is made to the MALSI TOF mass spectrometry target plate where typically a burst of 1000 droplets of the sample is spotted onto the MALDI plate. By this approach multi-layer depositions are achieved resulting in protein signal amplification in the MALDI instrument. This micro-tech principle provide high quality data from samples in the picomolar range. The built-in force feed-back control also further ensures precise and robust integration and interfacing of solid phase chip enrichment and piezodispencing technology. The micro-proteomic platform was compared to corresponding commercial preparation protocols, showing higher MS signal intensities for peptides generated from the resulting 2D-gel spots.
LC-MALDI-MS/MS: Reversed-phase chromatography was performed with an Agilent nano 1100 HPLC system (Agilent Technologies, Waldbronn, Germany). The HPLC effluent was directly fractionated onto a 144 position ABI MALDI target plate using an Agilent micro fraction collector and spots were deposited every 30 seconds during the gradient (90 spots/run). The spots were allowed to dry completely prior to addition of I μL of CHCA matrix. The MS/MS data from the MALDI-TOF/TOF instrument was acquired acording to a method previously described by Zhen et al. (J. Am. Soc. Mass Spectrom. 2004, 15, 803-822). A standard peptide mixture containing 6 peptides diluted to about 500 fmol/μL were applied on the six calibration spot positions of the target plate and used as external calibration points.
Protein identification by database searching: MS/MS data obtained from the MALDI- TOF/TOF instrument were searched using Mascot as the search engine. All searches were performed against the human, rat, and mouse subset of in-house protein sequence databases (Genseq P, RefSeqP, PDB, PIR, SwissProt and TREMBL). The Global Proteome Server (GPS) was used for submitting data acquired from the TOF/TOF for database searching. The Mascot searching was performed using the default settings for the TOF/TOF instrument as supplied by Matrix Science (peptide mass tolerance of 50 ppm and a fragment mass tolerance of 0.2 Da). Oxidation (M) and carbamidomethyl were allowed as variable modifications.
Results In total, 15 PPS samples and 43 OND/HC samples were included in the study. The patient samples originate from a larger study where the major focus was unbiased identification of biomarkers in Multiple sclerosis. PPS patient samples were included to serve as reference disease material together with controls OND/HC. We analysed in total 1499 2D-gel separated protein spots within the large study and 5-13 protein spots with very high predictive power for the PPS group were observed, that we then tested on a separate set of OND and PPS samples
The predictive modelling was carried out in two steps. First, the dataset of 1499 spots that contained 36 OND/HC and 9 PPS samples was used to select the spots with the highest predictivity and optimise model parameters. This set of samples will be referred to as the model set. The model optimisation and spot selection was done through a crossvalidation/bootstrapping procedure described more in detail below. In the second step, the predictivity of the model and the associated selected 13 spots was evaluated using a new set of samples, a test set, consisting of 7 OND and 6 PPS samples. See Figure 1 for an illustration of the datasets and how they were used in the analysis.
Further, prior to performing the test set validation, the gels for the samples used to build the model were subjected to a second round of matching for the selected 13 spots, and missing values can now be considered to arise from that they are below the detection limit.
Predictive models were built with an aim to identify a minimum number of markers that would give as good predictive value as possible. To this end, a variable selection procedure was coupled with a procedure for evaluating predictive performance. The latter is important to avoid over-fitting the data. The procedure works as follows:
A model was built on the training data, variable importance weights (VIP) calculated and the test set was predicted on the model. The percentage correctly classified samples was then recorded and new models were built on successively reduced numbers of variables as ranked by the VIP parameter. This process was then repeated for 100 random partitions of the samples into training and test sets, for models with a varying number of PLS components (1-10). Prior to building a model, the training set was standardized to zero mean and unit variance. The scaling parameters obtained from the training set were then applied to the test set prior to performing the prediction.
The model optimization was performed on two datasets, resulting from two different approaches to handling missing values (see materials & methods). In one dataset, missing values were considered to arise from that the protein levels were below the detection limit, and were replaced by a low value. In the other dataset, missing values were replaced using
the LS impute algorithm The PLS models with one component gave the best results for both datasets, and the prediction rate remained relatively constant as variables were dropped and it was seen that about 15-5 variables was sufficient to achieve a good prediction rate.
Variable ranks from the two models were combined to form an overal variable importance rank, and the top 13 variables were chosen for the final model. The corresponding protein spots were cut out from 2D-gels and identified by masspectrometry (Table 1) as described above. A ROC analysis was performed to find the cutoff that gave a balanced prediction rate, i.e. the proportion correctly classified samples is equal between the two classes. This resulted in a y cutoff at 0.35 for the model using the background- value replacement for missing values and 0.5 for the dataset that was imputed using LS Impute. The average prediction rate in the crossvalidation/bootstrap procedure was 90% and 99% correctly predicted samples for the two datasets, respectively.
A representative 2D-gel is shown in figure 3 and the locations of top- 13 protein spots are zoomed in. Each spot is given a unique database SSP number by the PDQuest software. Table 1 show protein identities obtained by mass spectrometry analysis and estimated molecular weight/isoelectric point.
Table 1. Summary Mass Spectrometry and database searching
To perform a validation of the model using the test set consisting of the new samples, a PLS model was built on the set of samples that were in the first set of analysis. The training set contained 9 PPS samples and 36 OND/HC samples and the test set contained 6 new PPS samples and 7 new OND samples. The parameters were selected as described above: 1 PLS component and the cutoff was chosen to be between 0.35 and 0.5 (based on the cutoffs obtained in the model optimization). Prior to building the model, the dataset had gone through a second matching round for the selected 13 spots, and missing values can now be considered to arise from that they are below the detection limit. These values were
10 set to a low value (2 ppm) representing the detection limit, and data were then log transformed.
Table 2 shows the VIP, VIP rank and regression coefficients for the complete and the training datasets. The values based on the complete dataset are similar to those for the is training dataset, however the rank for some of the spots in the middle range has been changed. This indicates the differences in VIP between these spots are minor, and that too much confidence cannot be placed in a specific rank number. However, the two top and the two bottom ranks are the same between the two datasets.
20 The top 11 -ranked protein based on VIP values are represented by only three proteins; gelsolin, kallikrein 6 and hemopexin. It should be stressed that one need all proteins together to obtain maximal prediction. PAM and GSHB scores the lowest values and it is likely these are of minor importance for the model. A type of sensitivity analysis was
carried out in which variables were dropped successively one variable at a time (based on VIP rank, Table 3), models built on the training dataset and recording the prediction rate for the test set. The prediction rate as a function of the number of variables (at cutoff 0.425) showed to be stable down to using just one variable for the OND/HC class, and down to 5 variables for the PPS class. At 4-1 variables the PPS class prediction drops from 6/6 to 5/6. Thus, the prediction is stable in terms of retaining the predictive ability when variables are dropped from the models.
Gelsolin is represented by fragments only, indicating that the quantity of full-length variant of gelsolin do not add value in a predictive model. Hemopexin show on the other hand a clear decrease of the full-length variant in parallel with an increase of fragmented hemopexin. The kallikrein 6 protein spot is likely to be a full-length variant (according to observed versus theoretical molecular weight).
Table 2. VIP and regression coefficients derived based on the complete (training samples+new samples) 13-variable data set and the training 13-variable data set respectively (log ppm).
Spot Protein ID VIP VIP rank Reg. coeff. VIP VIP rank Reg. coeff. number complete complete complete data training training set training set
(SSP) data data set
3408 GSN Fragment 1.30 1 0.053 1.24 1 0.048
3403 GSN Fragment 1.15 2 0.047 1.17 2 0.045
8221 KLK6 1.11 3 0.045 1.06 4 0.041
6605 HPX 1.09 4 -0.045 1.06 5 -0.041
8206 HPX Fragment 1.05 5 0.043 0.94 11 0.036
7208 HPX Fragment 1.03 6 0.042 1.00 8 0.039
5617 HPX 1.02 7 -0.041 1.11 -0.043
6611 HPX 0.99 8 -0.040 1.00 9 -0.039
7328 HPX Fragment 0.98 9 0.040 0.96 10 0.037
5631 HPX 0.98 10 -0.040 1.05 6 -0.040
5632 HPX 0.93 11 -0.038 1.04 7 -0.040
4414 PAM 0.78 12 -0.032 0.61 13 -0.023
7310 GSHB 0.53 13 0.022 0.73 12 0.028
Hierarchical clustering of all samples and proteins
To provide an overview of the samples in relation to the spots, two-dimensional hierarchical clustering was carried out. Hierarchical clustering is an unsupervised method, which allows data to selforganise according to similarity. A clear separation of the two patient groups was observed, and all samples except one (# 23) cluster within the correct diagnostic group. This is the same sample that was incorrectly classified in the PLS model. Interestingly, sample #23 originate from the only patient diagnosed with fibromyalgia, which indicate that the top- 13 identified protein described above may be used as biomarkers for this disease.
Thus, in a another aspect of the invention, there is provided a method for screening or diagnosis of fibromyalgia in a subject, for determining the stage or severity of fibromyalgia in a subject, for identifying a subject at risk of developing fibromyalgia, or for monitoring the effect of therapy administered to a subject having fibromyalgia, said method comprising:
(a) analysing a test sample of body fluid from the subject by two-dimensional electrophoresis to generate a two-dimensional array of features, said array comprising one
or more of the following protiens: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB; and
(b) comparing the abundance of the one or more PPS Features (PPSFs) in the test sample with the abundance of the one or more of said proteins in a body fluid sample from one or more subjects free from fibromyalgia, or with a previously determined reference range for that feature in subjects free from fibromyalgia, or with the abundance at least one Expression Reference Feature (ERF) in the test sample.
In one embodiment of this aspect, said array comprises one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
In a another aspect of the invention, there is provided a method for screening or diagnosis of fibromyalgia in a subject, for determining the stage or severity of fibromyalgia in a subject, for identifying a subject at risk of developing fibromyalgia, or for monitoring the effect of therapy administered to a subject having fibromyalgia, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following proteins: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB.
In one embodiment of this aspect, said method comprises quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
In another embodiment of this aspect, said body fluid is cerebrospinal fluid (CSF).
In another embodiment of this aspect, said body fluid is plasma.
In another embodiment of this aspect, said body fluid is serum. In another embodiment of this aspect, said body fluid is urine.
Fig 3a. Box-plots showing expression levels in ppm for 43 OND/HC and 15 PPS samples. The boxes are defined by the 25th and 75th percentile, thus 50 percent of the data is represented within each box. Upper and lower adjacent values, are represented by whiskers (defined by Spotfire™).
Table 3 a and b show mean, standard deviations, coefficent of variation (CV%) within the OND/HC and PPS groups, as well as the fold change, calculated as the ratio of the mean levels.
Table 3a. Mean, standard deviations, coefficent of variation (CV%) within the OND/HC and PPS groups, as well as the fold change, calculated as the ratio of the mean levels.
SSP ID Mean PPS Mean StdDev StdDev CV% PPS CV % Fold
OND/HC PPS OND/HC OND/HC change
3403 GSN Frag 754 331 266 118 35 36 2.28
3408 GSN Frag 1100 440 325 133 30 30 2.50
7310 GSHB_HUMAN 63 32 47 33 75 101 1.93
5617 HPX 2234 4835 1100 1598 49 33 -2.16
5631 HPX 2336 4949 1124 1786 48 36 -2.12
5632 HPX 2653 5128 1701 1837 64 36 -1.93
6605 HPX 807 2700 592 1625 73 60 -3.35
6611 HPX 144 1157 142 1532 98 132 -8.02
7208 HPX Frag 186 37 70 39 38 105 5.03
7328 HPX Frag 249 62 110 72 44 116 4.01
8206 HPX Frag 458 113 178 150 39 133 4.04
8221 KLK6 261 49 136 53 52 108 5.32
4414 PAM 43 148 42 93 97 63 -3.42
Table 3b
SSP ID Mean Mean StdDev StdDev CV % CV % Fol(
PPS OND/HC PPS OND/HC PPS OND/HC cha
3403_3408 GSN Frag 1854 771 505 234 27 30 2.4I
5617_5631_5632_6605_ _6611 HPX 8176 18768 4034 6230 49 33 -2.2
7208 7328 8206 HPX Frag 893 212 321 183 36 86 4.2
The results represent a totally new approach to understand the PPS. Earlier there are evidence of both an ongoing neurodegeneration and a chronic inflammation in the central nervous system of these patients. Here, we present a molecular analysis of the CSF that very specifically defines the Postpolio syndrome, in that a certain well defined group of proteins are differentially expressed in the CSF of these patients as compared to controls. Interestingly, the differentially expressed proteins, are previously described to be involved in a variety of inflammatory processes and in apoptosis. The findings may impact both the understanding of the disease process and a potential development of better diagnostic and prognostic markers of PPS
Gelsolin- fragments are the most prominent proteins present in the CSF of persons with PPS. Gelsolin is tightly associated with apoptosis, and both pro-apoptotic and anti- apoptotic roles have been described. As to the pro-apoptotic pathaway gelsolin interacts with the caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases which in turn has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severedactin filaments in vitro in a Ca2+-independent manner. Expression of the gelsolincleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis. As to the antiapoptotic pathway, Gelsolin can prevent apoptosis via the mitochondrial pathway (Kusano H et al Oncogene, 2000, 19,4807). Futhermore, in vivo experiments in gelsolin gene deleted mice demonstrated that the defect mice developed much more apoptosis of liver cells than wild type mice (Leifeld L et al AmJ Pathol, 2006). In PPS, there mere increased expression naturally cannot discriminate in between any pro- or anti apoptic role. In the first case, it could represent an inherent defence mechanism against intrathecal noxious events, in the second, it could be the consequence of such events. In any case, the increased protein expression suggests an increased apoptotic activity intrathecally in PPS.
In this study, we observe a decrease of the full-length hemopexin protein, along with increased levels of several hemopexin- fragments in CSF. The reasons for an increased cleavage of hemopexin in PPS is unclear, but suggest that there is an increased proteolytic cleavage of the protein. We observe increased levels of the serine protease kallikrein-6, which could be a potential candidate for the observed fragmentation of hemopexin. However, in silico examinations of possible proteases using protease-databases (e.g. Merops
did not reveal any previously known protease cleavage site except E. coli peptidase S26.
Hemopexin, is a 80-Kda glycoprotein mainly expressed in the liver and it belongs to the family of acute-phase proteins induced during inflammatory events (Melchers W, de Visser M, Jongen P, et al. The postpolio syndrome: no evidence for poliovirus persistence. Ann Neurol 1992;32(6):728-32]). The most well characterized function of hemopexin is to bind heme and to protect against oxidative stress by receptor-mediated induction of heme oxygenase (Jubelt B, Salazar-Grueso EF, Roos RP, Cashman NR. Antibody titer to the poliovirus in blood and cerebrospinal fluid of patients with post-polio syndrome. Ann N Y Acad Sci 1995;753:201-7). Interestingly, the role of hemopexin seems to be different in CNS as compared to the systemic functions. Experiments have shown that hemopexin can be induced in response to tissue damage in CNS, mainly by Schwann cells but also by fibroblasts and macrophages, and that this expression of hemopexin may be induced by IL- 6 (Madore, N., Camborieux, L., Bertrand, N. & Swerts, J.P. Regulation of hemopexin synthesis in degenerating and regenerating rat sciatic nerve. J Neurochem 72, 708-15 (1999). In Minimal Change Disease (MCD) there is a change in hemopexin expression pattern. A comparison between MCD and PPS, reveals common features like, the lack of inflammatory findings, response to pharmacological treatment (corticosteroids and IVIG respectively) and interestingly decreased hemopexin level of full-length hemopexin. Moreover, in MCD there is an activation of hemopexin proteolytic activity resulting in degradation of extra cellular matrix (Bakker, W. W. et al. Altered activity of plasma hemopexin in patients with minimal change disease in relapse. Pediatr Nephrol 20, 1410-5 (2005)). Although this event occurs in the kidney in MCD, the function for HPX may be
similar in CNS. Hypothetically, the proteolytic activation of hemopexin may result in tissue damage, which in turn cause an activation of TNF-α and eventually apoptosis.
The origin to the fragmentation of hemopexin in PPS is unknown and remains to investigated further. If the fragmentation will contribute to loss or gain of function of hemopexin is not clear but it is most likely that it have consequences for the pathogenesis in PPS. One possibility is that the fragmentation and loss of hemopexin function in the CNS of persons with PPS could be negative in that protection of ECM is lost as well as the suggested protection against oxidative stress.
Kallikrein 6 was the third most prominently altered protein in the CSF of persons with PPS. There was an approximate threefold increase of its levels in PPS as compared to both OND and SPMS. In addition to this single kallikrein 6 spot (SSP 8221) we identified one other spot (SSP8209) as kallikrein 6 (data not shown), although at lower prediction scores (VIP rank #35). However, both kallikrein 6 spots showed increased levels in PPS, which support the possible impact of kallikrein 6 in PPS. Kallikrein 6 is a serine protease belonging to a gene family with 15 members and is preferentially expressed in the CNS and at highest levels in the spinal cord and brain stem (Diamandis, E. P., Yousef, G. M., Petraki, C. & Soosaipillai, A.R. Human kallikrein 6 as a biomarker of alzheimer's disease. Clin Biochem 33, 663-7 (2000)). It has a trypsin-like broad substrate enzymatic activity (Scarisbrick, LA. et al. Potential scope of action of tissue kallikreins in CNS immune- mediated disease. J Neuroimmunol 178, 167-76 (2006); Grimby, G., Stalberg, E., Sandberg, A. & Sunnerhagen, K.S. An 8-year longitudinal study of muscle strength, muscle fiber size, and dynamic electromyogram in individuals with late polio. Muscle Nerve 21, 1428-37 (1998)). Kallikrein 6 is expressed in neurons, oligodendrocytes, and strongly up-regulated in astrocytes, macrophages/microglia in CNS pathologies such as spinal cord trauma and in human or experimental inflammatory demyelinating disease. Hence, its up-regulation can be regarded as a part of a CNS innate immunity response to a variety of insults, both traumatic and primary inflammatory events. It has also been detected in the CSF/CNS of neurodegenerative diseases such as Alzheimers and
Parkinsons disease (Diamandis EP, Yousef GM, Petraki C, Soosaipillai AR. Human kallikrein 6 as a biomarker of alzheimer's disease. Clin Biochem. 2000 Nov;33(8):663-7.
PMID: 11166014). In view of previous data on the enzyme its increase in the CSF of persons with PPS our finding provides a strong further argument for an ongoing nervous tissue damaging process in the syndrome. Since kallikrein 6 was detected as a full-length protein and is accessible for measurements with ELISA it constitutes an attractive candidate as biomarker in PPS to be validated in larger materials. Additional determinations of human kallikrein 6 (hK6) levels were assessed in new samples of CSF (PPS, n=20 and OND, n=20) using a commercial hK6 ELISA kit (IBEX Pharmaceuticals, Inc., Montreal, QC, Canada). Differences in the kallikrein 6 levels in CSF were tested for significance with the non-parametric Mann Whitney test, which showed that the increased expression of kallikrein 6 in PPS was confirmed by this method (P =0.0005). The driving force of it induction in PPS remain unclear, but in view of its induction in lesions of primary CNS inflammatory diseases, we speculate that proinflammatory cytokines such as TNF and interferon gamma may be considered, in particular in view of their known increased production in PPS. The functional consequences of the increased Kallikrein 6 levels in PPS are unclear. As with most inflammatory mediator and effector molecules, both beneficial and detrimental effects can be considered. On the potential beneficial side, the enzyme modifies the conditions for neurite outgrowth after spinal cord trauma, it has been suggested to protect against amyloid deposition in Alzheimers disease. On the detrimental side, the enzyme mediates toxicity against myelin/oligodendrocytes in experimental autoimmune encephalomyelitis.
The increased differential expression of hemopexin, gelsolin and kallikrein-6, involved in apoptosis and inflammatory damage lends strong support to the idea that the continuous worsening of symptoms in PPS is an active neurodegenerative process, possibly driven by local inflammation. This argues against the idea that the worsening would be due to normal ageing, on top of a paucity of remaining motor units. For reference, we have also analysed an age-matched group of patients diagnosed with secondary progressive multiple sclerosis (SPMS). However, the protein pattern in PPS differed distinctly from that in SPMS, which is a slow, but different neurodegenerative disease probably driven by chronic inflammation, though with potentially very different driving forces.
The invention described in detail provides methods and compositions for clinical screening and diagnosis of Postpolio Syndrome (PPS), in a mammalian subject for identifying patients most likely to respond to a particular therapeutic treatment, for monitoring the results of PPS therapy, for drug screening and drug development. The invention also encompasses the administration of therapeutic compositions to a mammalian subject to treat or prevent PPS. The mammalian subject may be a non-human mammal, but is preferably human.
For clarity of disclosure, and not by way of limitation, the invention will be described with respect to the analysis of CSF samples. However, as one skilled in the art will appreciate, the assays and techniques described below can be applied to other types of samples, including a body fluid (for example but without limitation: blood, serum, plasma, saliva or urine), a tissue sample from a subject at risk of having or developing PPS or homogenate thereof. The methods and compositions of the present invention are useful for screening and diagnosis of a living subject, but may also be used for postmortem diagnosis in a subject, for example, to identify family members of the subject who are at risk of developing the same disease.
The following definitions are provided to assist in the review of the instant disclosure.
"Feature" refers to a spot identified in a 2D gel, and the term "Postpolio Syndrome feature (PPSF) refers to a feature that is differentially expressed in a first sample or sample set from a subject having PPS compared with a second sample or sample set from a subject free from PPS. A feature or spot identified in a 2D gel is characterized by its isoelectric point (pi) and apparent molecular weight (MW) as determined by 2D gel electrophoresis. As used herein, a feature is "differentially expressed" in a first sample or sample set with respect to a second sample or sample set when a method for detecting the said feature (e.g., 2D electrophoresis) gives a different signal when applied to the first and second samples or sample sets. Particularly, the relative abundance of a feature in the two samples or sample sets is determined in reference to its normalized signal, in two steps. First, the signal obtained upon detecting the feature in a first sample or sample set is normalized by reference to a suitable background parameter, e.g., (a) to the total protein in the sample
being analyzed (e.g., total protein loaded onto a gel); (b) to an Expression Reference Feature (ERF) i.e., a feature whose abundance is substantially invariant, in the population of subjects being examined. Secondly, the normalized signal for the feature in the first sample or sample set is compared with the normalized signal for the same feature in the second sample or sample set in order to identify features that are "differentially expressed" in the first sample or sample set with respect to the second sample or sample set.
"Variant" as used herein refers to a polypeptide which is a member of a family of polypeptides that are encoded by a single gene or from a gene sequence within a family of related genes and which differ in their pi or MW, or both. Such variants can differ in their amino acid composition (e. g. as a result of alternative mRNA or premRNA processing, e.g. alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from 10 differential post-translational modification (e.g., glycosylation, acylation, phosphorylation) .
"PPSF analog" refers to a polypeptide that possesses similar or identical function(s) as a PPSF but need not necessarily comprise an amino acid sequence that is similar or identical to the amino acid sequence of the PPSF, or possess a structure that is similar or identical to that of the PPSF. As used herein, an amino acid sequence of a polypeptide is "similar" to that of a PPSF if it satisfies at least one of the following criteria: (a) the polypeptide has an amino acid sequence that is at least 30 To (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of the PPSF; (b) the polypeptide is encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding at least 5 amino acid residues (more preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, 30 or at least 150 amino acid residues) of the PPSF; or (c) the polypeptide is encoded by a nucleotide sequence that is at least 30% (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95% or at least 99%) identical to the nucleotide sequence encoding the PPSF. As used herein, a polypeptide with "similar structure" to that of 35 a PPSF refers to a polypeptide that has a similar secondary, tertiary or Quarternary structure as that of the PPSF. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, mass spectrometry and crystallographic electron microscopy.
"Chimeric Antibody" refers to a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a marine mAb. (See, e.g., Cabilly et al., U.S. Patent No. 10 4,816,567; and Boss et al., U.S. Patent No. 4,816397, which are incorporated herein by reference in their entirety.)). For example, a portion of the antibody may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHl, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric antibodies.
"Humanised Antibody" refers to a molecule from non-human species having one or 15 more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
"Derivative" refers to a polypeptide that comprises an amino acid sequence of a second polypeptide that has been altered by the introduction of at least one amino acid residue substitution, deletion or addition. The derivative polypeptide possesses a similar or identical function as the second polypeptide.
"Fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid 25 residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a second polypeptide. Prefereably, but not necessarily, the fragment of a PPSF possesses the functional activity of the PPSF.
The "percent identity" of two amino acid sequences or of two nucleic acid sequences can be or is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in either sequences for best alignment with the other sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences that results in the highest percent identity. The 35 percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = # of identical positions/total # of positions x 100). The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a 40 mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. MoI. Biol. 215:403-410 have incorporated such an algorithm.
BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score = SO, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be 5 utilized as described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Another example of a mathematical algorithm utilized for the comparison of sequences 10 is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within 15 FASTA, ktup is a control option that sets the sensitivity and speed of the search.
"Diagnosis" refers to diagnosis, prognosis, monitoring, characterizing, selecting patients, including participants in clinical trials, and identifying patients at risk for or having a particular disorder or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment.
"Treatment" refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure the infirmity or malady in the instance where the patient is afflicted.
"Agent" refers to all materials that may be used to prepare pharmaceutical and diagnostic compositions, or that may be compounds, agonists, antagonists, nucleic acids, polypeptides, fragments, isoforms, variants, or other materials that may be used independently for such purposes, all in accordance with the present invention.
"Cerebrospinal fluid (CSF)" refers to the fluid that surrounds the bulk of the CNS, as described in Physiological Basis of Medical Practice (J.B. West, ea., Williams and Wilkins, Baltimore, MD 1985). CSF includes ventricular CSF and lumbar CSF.
"Serum" refers to the supernatant fluid produced by clotting and centrifugal 40 sedimentation of a blood sample.
"Plasma" refers to the supernatant fluid produced by inhibition of clotting (for example, by citrate or EDTA) and centrifugal sedimentation of a blood sample.
"Blood" as used herein includes serum and plasma.
"Two-dimensional electrophoresis" (2D -electrophoresis) means a technique comprising isoelectric focusing, followed by denaturing electrophoresis; this generates a two- dimensional gel (2D-gel) containing a plurality of separated proteins.
In one aspect of the invention, two-dimensional electrophoresis is used to analyze CSF from a subject, preferably a living subject, in order to detect or quantify the expression of one or more Postpolio Syndrome Features (PPSFs), screening, prevention or diagnosis of PPS, to determine the prognosis of a subject having PPS, to monitor progression of PPS, to
monitor the effectiveness of PPS therapy, for identifying patients most likely to respond to a particular therapeutic treatment, or for drug development.
By way of example and not of limitation, a number of samples from subjects having PPS and samples from subjects free from PPS are separated by two-dimensional electrophoresis, and the fluorescent digital images of the resulting gels are matched to a chosen representative primary master gel image. This process allows any gel feature, characterized by its pi and MW, to be identified and examined on any gel of the study.
In particular, the amount of protein present in a given feature can be measured in each gel; this feature abundance can be averaged amongst gels from similar samples (e.g. gels from samples from subjects having PPS). Finally, statistical analyses can be conducted on the thus created sample sets, in order to compare 2 or more sample sets to each other.
Claims
1. A method for screening or diagnosis of Postpolio Syndrome (PPS) in a subject, for determining the stage or severity of PPS in a subject, for identifying a subject at risk of developing PPS, or for monitoring the effect of therapy administered to a subject having PPS, said method comprising:
(a) analysing a test sample of body fluid from the subject by two-dimensional electrophoresis to generate a two-dimensional array of features, said array comprising one or more of the following protiens: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB; and
(b) comparing the abundance of the one or more PPS Features (PPSFs) in the test sample with the abundance of the one or more of said proteins in a body fluid sample from one or more subjects free from PPS, or with a previously determined reference range for that feature in subjects free from PPS, or with the abundance at least one Expression Reference Feature (ERF) in the test sample.
2. The method according to claim 1, wherein said array comprises one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
3. A method for screening or diagnosis of Postpolio Syndrome (PPS) in a subject, for determining the stage or severity of PPS in a subject, for identifying a subject at risk of developing PPS, or for monitoring the effect of therapy administered to a subject having PPS, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following proteins: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB.
4. The method according to claim 3, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
5. The method according to any one of claims 1 to 4, wherein the body fluid is cerebrospinal fluid (CSF).
6. The method according to any one of claims 1 to 4, wherein the body fluid is plasma.
7. The method according to any one of claims 1 to 4, wherein the body fluid is serum.
8. The method according to any one of claims 1 to 4, wherein the body fluid is urine.
9. An antibody capable of immunospecific binding to a Postpolio Syndrome Feature (PPSF) according to claim 2 or 4.
10. An antibody according to claim 9, wherein the antibody is a monoclonal antibody.
11. An antibody according to claim 9 or 10, wherein the antibody is selected from a chimeric antibody, bispecific antibody and a humanised antibody.
12. A kit comprising one or more antibodies as claimed in any one of claims 9 to 11 and/or one or more PPSF as defined in claim 2 or 4, other reagents and instructions for use.
13. The kit of claim 12 for use in the screening or diagnosis of Postpolio Syndrome in a subject, for determining the stage or severity of Postpolio Syndrome in a subject, for identifying a subject at risk of developing Postpolio Syndrome, or for monitoring the effect of therapy administered to a subject having Postpolio Syndrome.
14. The kit according to claim 12 or 13 comprising a plurality of antibodies as claimed in any one of claims 9 to 13 and/or a plurality of PPSFs as defined in claim 2 or 4.
15. A pharmaceutical composition comprising a therapeutically effective amount of an antibody, or a fragment or derivative of an antibody according to any one of claims 9 to 11 and a pharmaceutically acceptable carrier.
16. A method of treating or preventing Postpolio Syndrome comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody according to any one of claims 9 to 11.
17. A method of screening for agents that interact with one or more Postpolio Syndrome features (PPSFs) as defined in claim 2 or 4, said method comprising:
(a) contacting an PPSF with a candidate agent; and
(b) determining whether or not the candidate agent interacts with the PPSF.
18. The method according to clain 17, wherein the determination of interaction between the candidate agent and the PPSF comprises quantitatively detecting binding of the candidate agent and the PPSF.
19. A method of screening for or identifying agents that modulate the expression or activity of one or more more Postpolio Syndrome features (PPSFs), comprising:
(a) contacting a first population of cells expressing the PPSF with a candidate agent;
(b) contacting a second population of cells expressing said PPSF with a control agent; and
(c) comparing the level of said PPSF(s) in the first and second populations of cells, or comparing the level of induction of a downstream effecter in the first and second populations of cells.
20. A method of screening for or identifying agents that modulate the expression or activity of one or more more Postpolio Syndrome features (PPSFs) as defined in claim 2 or 4, said method comprising:
(a) administering a candidate agent to a first mammal or group of mammals;
(b) administering a control agent to a second mammal or group of mammals; and
(c) comparing the level of expression of the PPSF, or mRNA encoding said PPSF in the first and second groups, or comparing the level of induction of a downstream effecter in the first and second groups.
21. The method according to any one of claims 17 to 19, wherein administration of a candidate agent results in an increase in the level of said PPSF(s), or mRNA encoding said PPSF(s), or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
22. The method according to any one of claims 17 to 19, wherein administration of a candidate agent results in a decrease in the level of said PPSF(S), or mRNA encoding said PPSF(s), or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
23. A method of screening for or identifying agents that modulate the activity of one or more of the more Postpolio Syndrome features (PPSFs) as defined in claim 2 or 4, said method comprising:
(a) in a first aliquot, contacting a candidate agent with the PPSF(s) and;
(b) determining and comparing the activity of the PPSF(s) in the first aliquot after addition of the candidate agent with the activity of PPSF(s) in a control aliquot, or with a previously determined reference range.
24. The method according to any one of claims 17 to 23, wherein the PPSF(s) is a recombinant protein.
25. The method according to any one of claims 17 to 23, wherein the PPSF(s) is immobilized on a solid phase.
26. A method for screening or diagnosis of Postpolio Syndrome in a subject or for monitoring the effect of an anti-Postpolio Syndrome drug or therapy administered to a subject, comprising:
(a) contacting at least one oligonucleotide probe comprising 10 or more consecutive nucleotides complementary to a nucleotide sequence encoding an PPSF as defined in claim 2 with RNA obtained from a biological sample from the subject or with cDNA copied from the RNA wherein said contacting occurs under conditions that permit hybridization of the probe to the nucleotide sequence if present;
(b) detecting hybridization, if any, between the probe and the nucleotide sequence; and
(c) comparing the hybridization, if any, detected in step (b) with the hybridization detected in a control sample, or with a previously determined reference range.
27. The method as claimed in claim 26, wherein step (a) includes the step of hybridizing the nucleotide sequence to a DNA array, wherein one or more members of the array are the probes complementary to a plurality of nucleotide sequences encoding distinct PPSFs.
28. A method of treating or preventing Postpolio Syndrome comprising administering to a subject in need of such treatment or prevention a therapeutically effetive dose of an agent that modulates the activity of one or more of more Postpolio Syndrome features (PPSFs) as defined in claim 2 or 4; whereby the symptoms of Postpolio Syndrome are ameliorated.
29. A method for screening or diagnosis of fibromyalgia in a subject, for determining the stage or severity of fibromyalgia in a subject, for identifying a subject at risk of developing fibromyalgia, or for monitoring the effect of therapy administered to a subject having fibromyalgia, said method comprising:
(a) analysing a test sample of body fluid from the subject by two-dimensional electrophoresis to generate a two-dimensional array of features, said array comprising one or more of the following protiens: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB; and
(b) comparing the abundance of the one or more PPS Features (PPSFs) in the test sample with the abundance of the one or more of said proteins in a body fluid sample from one or more subjects free from fibromyalgia, or with a previously determined reference range for that feature in subjects free from fibromyalgia, or with the abundance at least one Expression Reference Feature (ERF) in the test sample.
30. The method according to claim 29, wherein said array comprises one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
31. A method for screening or diagnosis of fibromyalgia in a subject, for determining the stage or severity of fibromyalgia in a subject, for identifying a subject at risk of developing fibromyalgia, or for monitoring the effect of therapy administered to a subject having fibromyalgia, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following proteins: gelsolin or fragments thereof; kallikrein 6; hemopexin or fragments thereof; PAM; and GSHB.
32. The method according to claim 31, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following PPS Features (PPSFs): gelsolin fragments 3408 and 3403; kallikrein 6; hemopexin 6605; hemopexin 5617; hemopexin 6611; hemopexin 5631; hemopexin 5632; hemopexin fragment 8206; hemopexin fragment 7208; hemopexin fragment 7328; PAM; and GSHB.
33. The method according to any one of claims 29 to 32, wherein the body fluid is cerebrospinal fluid (CSF).
34. The method according to any one of claims 29 to 32, wherein the body fluid is plasma.
35. The method according to any one of claims 29 to 32, wherein the body fluid is serum.
36. The method according to any one of claims 29 to 32, wherein the body fluid is urine.
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