WO2005091969A2 - Marqueurs pre-symptomatiques pour des maladies associees a des encephalopathies spongiformes transmissibles (ste) - Google Patents

Marqueurs pre-symptomatiques pour des maladies associees a des encephalopathies spongiformes transmissibles (ste) Download PDF

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WO2005091969A2
WO2005091969A2 PCT/US2005/007189 US2005007189W WO2005091969A2 WO 2005091969 A2 WO2005091969 A2 WO 2005091969A2 US 2005007189 W US2005007189 W US 2005007189W WO 2005091969 A2 WO2005091969 A2 WO 2005091969A2
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cells
marker
level
sample
tse
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Laura Manuelidis
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Laura Manuelidis
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/7056—Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
    • G01N2333/70564—Selectins, e.g. CD62
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • the invention relates to methods to assess the presence of infection that affect the brain and can be associated with abnormal amyloid components, such as Creutzfeldt- Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, and other related diseases associated with host-encoded prion protein (PrP) amyloid and to distinguish them from other such conditions that are not the result of infection, such as Alzheimer's. More specifically, the invention provides markers for the conditions that may be assessed prior to symptoms previously recognized.
  • the ability of the SY strain to protect against infection by the FU strain was ascribed to a successful immune response (ibid . It has been shown that the infectious particle for CJD has a viral size of about 25 nm and infectivity is markedly reduced by conditions that disrupt viral core components.
  • abnormal PrP is formed in the brain about 90 days after infection, i.e., experimental inoculation with rodent-passed CJD or similar CJD strains. Thus, at the time the markers enumerated above were determined, the abnormal PrP presence in the brain was well established.
  • the markers described in the present invention are present at detectable levels before the appearance of abnormal PrP. Furthermore, the markers appear not only in the microglia, but also in the peripheral system, including blood, gut, urine and the like, and in cells isolated from these sources.
  • these markers provide a convenient early warning assay for TSE infection prior to the appearance of alternative symptomologies, including the appearance of PrP in the microglia. Because these markers are present in the peripheral system, the assessment can be made on accessible body fluids and cells. Further, because some of the markers are different from those in Alzheimer's disease brain, they can be used to distinguish TSE infection from Alzheimer's in symptomatic patients. In addition, use of the markers may distinguish various TSE strains.
  • the invention provides markers for diagnosis of early stage TSE infection, for distinguishing TSE from Alzheimer's, and for identifying TSE strains.
  • the markers can be assessed in the brain or in the peripheral system and in isolated cell preparations and can determine the presence of TSE infection prior even to the formation of abnormal prion protein (PrP).
  • PrP abnormal prion protein
  • the pattern of markers is also indicative of the stage of infection.
  • the invention is directed to a method to identify a subject that has been infected with a transmissible spongiform encephalopathy (TSE) which method comprises determining, in at least one sample, e.g., body fluid or isolated cell preparation of said subject, the level of a marker associated with early response to infection and comparing the level to that characteristic of a corresponding sample from a normal subject. A difference in level of the marker as compared to the level in normal samples identifies the subject as infected. Multiple markers and patterns of marking can also be determined.
  • TSE transmissible spongiform encephalopathy
  • Suitable early markers include, for example, serum amyloid A3 (SAA3), L-selectin, MlP-l ⁇ , MlP-l ⁇ , MCP, IL-l ⁇ , TNF ⁇ , and Toll-like receptor (TLR)4.
  • SAA3 serum amyloid A3
  • L-selectin L-selectin
  • MlP-l ⁇ MlP-l ⁇
  • MCP IL-l ⁇
  • TNF ⁇ Toll-like receptor
  • TLR Toll-like receptor
  • Early detection of infection can also be demonstrated using elevated levels of CD72, CD84, LY86, CXCL13, IFI204, LY9, IFI202, ImmResGl, CXCL10. RSG15, PRF7, RANTES and OAS as indicators.
  • the level of GAPDH may be used as a control. All of these genes provide distinct levels of mRNA and protein prior to formation of detectable levels of PrP.
  • markers are expressed at different levels depending on the nature of the sample assessed.
  • the expression levels of markers are determined in isolated cell types which appear to provide clear information as to the levels of the indicators.
  • individual cell types may be stimulated by treating with specific chemicals and inducers to accentuate differences in cells obtained from subjects exposed to TSE versus normal controls.
  • Cells obtained from TSE inoculated subjects appear to display accentuated differences in levels of expression of many of the marker genes when treated with general immune stimulants such as double-stranded RNA or other agents which interact with the Tolllike receptors.
  • one embodiment of the invention relates to determining levels of indicator genes in cell types from tissues and fluids peripheral to the central nervous system (CNS).
  • the invention is directed to a method to distinguish TSE infection from Alzheimer's, which method comprises determining levels of markers characteristic of TSE, but not associated with Alzheimer's.
  • the invention is directed to discriminating among various stages of the disease by assessing the levels of these markers as a function of time.
  • Figures 1A-1D show the level of marker mRNA in brain and microglia as determined by RT-PCR as a function of time.
  • Figures 1C-1D also demonstrate the enhancing effect of treating assessed microglia cells with poly I:C.
  • Figures 2A-2I show a graphical representation of the time course of expression in microglia for various genes in TSE infection.
  • the invention provides markers for TSE which are detectable and measurable prior even to the appearance of pathologic prion protein.
  • the assays can be conducted on tissue such as brain, gut or spleen tissue from a subject but most conveniently from biological fluids of the subject, such as cerebrospinal fluid, blood or its components, urine, lymphatic fluid, saliva or even contents of the gastric system.
  • the subject is generally a mammalian subject; as noted above, TSE conditions are found in zoo animals, cats, sheep, cattle, and humans and other primates.
  • Convenient fluids for assay include plasma and serum prepared as generally understood for diagnostic tests.
  • the assays are conducted on isolated cell types such as microglia, astrocytes, and various cells associated with the spleen such as B cells, T cells and myeloid cells.
  • isolated cell types such as microglia, astrocytes, and various cells associated with the spleen such as B cells, T cells and myeloid cells.
  • the samples useful for assessment of the level of gene expression either at the mRNA level or the protein level include tissue samples which comprise heterogeneous cell types, fluids which may themselves contain cells, and "isolated cell preparations" which are relatively homogeneous as to cell type.
  • Isolated cell preparations contain at least 80%, 85%, 90%, 95% or more than 95% cells of the same differentiated form and tissue origin, such as myeloid cells from the microglia, myeloid cells from the spleen, B cells from the spleen, B cells from plasma, and the like. Cells derived from tissues and fluids peripheral to the CNS are particularly convenient. [0022] The information obtained by assessing gene expression is evaluated by comparing these levels in the sample to be tested with the levels found in a corresponding normal sample.
  • corresponding normal sample is meant a sample prepared in substantially the same manner as that tested but from one or more individuals known not to be infected with a TSE agent.
  • the data with regard to normal levels may be obtained simultaneously using one or more corresponding normal samples as a control or, preferably, data obtained from normal samples can be stored and retrieved for comparison with the test results.
  • Levels in a "corresponding normal sample” thus is construed to refer to data associated with such samples however obtained and stored.
  • the markers can be assessed by a variety of methods, either by determining the level of messenger RNA transcribed by the expressed gene or by determining the level of protein produced or both. Typically, the level of mRNA or protein detectable in the sample will differ at least two-fold, or three-fold or five-fold, 10-fold, 20-fold or 40-fold from that in a corresponding normal sample.
  • the expression levels of the markers described herein are variable over the time period post inoculation and thus, depending on the nature of the markers chosen, and if applicable, the nature of the pattern obtained, the stage of progression of the TSE infection can be evaluated.
  • the levels of the markers assessed may increase or decrease and the levels in cells from TSE inoculated subjects are at least 2.5-fold higher or lower than those in normal cells.
  • markers may be useful to distinguish TSE infection from other conditions which affect the brain, such as Alzheimer's disease. Since the markers assessed herein are associated with response to infection, elevated levels of these markers in either early or late stage are not found in Alzheimer's subjects. [0024] In order to make a complete assessment, it may also be useful to obtain expression levels and/or patterns from a variety of sample types, such as B cells as well as astrocytes. Practitioners can readily design protocols which provide the maximum amount of diagnostic information in the most efficient manner by selecting those markers appropriate to the diagnostic purpose. Assessment of multiple markers in only a single sample type or in multiple sample types may also be employed. It may be advantageous to utilize markers that have different characteristics.
  • the patterns exhibited by markers such as GFAP, CD84, LY86, CXCL13, IFI204, LY9, and CD72 are similar and are biphasic in nature; weaker biphasic patterns (bridging both early and late stages of infection) are exhibited by ISG15, IRF7, IFN- ⁇ , PKR and IRF2. Fairly consistent increases in levels of expression as the condition progresses is exhibited by IFI202, ImmResGl, and CXCL10.
  • the marker is other than GFAP, CD84, LY86, CXCL13, IFI204, LY9, CD72, ISG15, IRF7, IFN- ⁇ , PKR, IRF2, IFI202, ImmResGl, or CXCL10.
  • ears stage or “early markers” refer to those whose levels are altered prior to 50 days subsequent to infection.
  • Middle stage markers are those where levels differ from those in normal samples between 50 and 80 days.
  • “Late stage markers” or “late markers” are those that are altered as compared to normal tissue during the period subsequent to 80 days after initial infection.
  • telomeres For assessing mRNA levels, a variety of methods is known, including Northern blot probed with an oligonucleotide which hybridizes under stringent conditions to the mRNA to be detected, use of quantitative PCR employing appropriate primers, use of other amplification techniques or by microarrays. Levels can be assessed using standard gene chip technology or other art-known methods.
  • fragments and modified forms that are immunospecific can be used as the detection reagent with suitable labels such as radioactive labels, fluorescent labels, enzyme labels and the like.
  • suitable labels such as radioactive labels, fluorescent labels, enzyme labels and the like.
  • suitable labels such as radioactive labels, fluorescent labels, enzyme labels and the like.
  • Microarrays designed to detect proteins are also available, and two-dimensional chromatography could also be employed. Flow cytometric techniques can also be used.
  • Expression products of the genes that are upregulated include expression products of serum amyloid A3 (SAA3), and L-selectin as well as MlP-l ⁇ , MIP-1 ⁇ , MCP1, IL-1 ⁇ , TNF ⁇ , IFI202, IFI204, CD72, LY9, CXCL10, and CX3CR1 and others as noted in the examples below.
  • SAA3 serum amyloid A3
  • L-selectin as well as MlP-l ⁇ , MIP-1 ⁇ , MCP1, IL-1 ⁇ , TNF ⁇ , IFI202, IFI204, CD72, LY9, CXCL10, and CX3CR1 and others as noted in the examples below.
  • Suitable oligonucleotides, PCR primers, and antibodies are either available in the art or can readily be prepared for these known expression products.
  • the examples provide experimental evidence of altered levels of markers associated with various stages of the infection and thus various combinations and sub-combinations of these markers can conveniently be used as the basis
  • markers include SAA3, CD 84, cathepsin S., or any of the markers shown in Table 1 below as indicative of early stage infection and combinations thereof.
  • MIP-1 ⁇ is also a convenient marker; it is present in late stage as well but because it is present in the early period, it can be used to detect the presence of infection of subjects who show no symptomology.
  • Elevated levels of CXCL13 are present at high levels in late stage infection but not early stage infection, and thus would be expected to correlate with clinical symptoms and to distinguish the presence of TSE infection from alternative diagnoses such as Alzheimer's.
  • Example 1 Determination of Upregulated Genes
  • CD-I mice were intracerebrally inoculated with 30 ⁇ l of a 1% brain homogenate containing the FU (fast acting) strain of the CJD agent. In parallel, animals were inoculated with normal brain homogenate to control for nonspecific effects of intracerebral inoculation.
  • TCCATGACGTTCCTGATGCT unmethylated CpG DNA oligonucleotides
  • TCCATGAGCTTCCTGATGCT oligonucleotides with an inverted CpG motif
  • Cathepsin L 65 20 CAGGGCCAGTGCGGGTCTTGTT GTTGTCCCGGTCTTTGGCTATTTTGATGTA
  • CD14 59 25 CTTCCTCAGATCCGAAGCCAGATTG TCGCCCAATTCAGGATTGTCAG
  • CD48 63 26 ACCACCGGCAGCAATGTAACCCTG GTCGTTCTTGCTGCTTACAGGATTGC
  • CD68 60 CAACAAAACCAAGGTCCAGGGA CCAATGATGAGAGGCAGCAAGA
  • Cystatin F 65 28 CAGCCATGTGGCTGGCCATTCTGCTTG ACTTCAGAGTAGCAATATAGAGTCCGC
  • L-selectin 65 31 ACTCTGGGAAATGGAACGATGAC AATGAAGAGGGGGTTGTAGTCACC
  • Properdin 72 25 AGAGACATCAGGGTAGAAGACTGCTG ATAGGCTGGTCCTGAGCAGGGTTTC
  • TLR2 68 25 ACAGTAGAGAACAGCAAGGTCTTCC GCTCTTGCAGCCGAGGCAAGAAC
  • T R3 65 28 GCAGTTTCCAACTCTGGATCTACC GTGTTTGCAAAGACATTTGAAAGGGTG
  • TLR4 68 25 TCGTTCAGTGAGCTACCACAGTTGC TGCCATTAGGCAGGGTGCCATTGG
  • TLR9 68 31 TTTCTCTTGGTTAGGCCAACTCAGG ACTGGGATTTCATCTAAGCCGTTGG
  • PCR products were separated on agarose gels, transferred to Biodyne B nylon membranes (Pierce, Rockford, IL), then visualized with the BrightStar ® BioDetectTM kit (Ambion, Austin, TX) and BioMaxTM MR film (Kodak, New Haven, CT). Densitometry was conducted using National Institutes of Health (NIH) Image, with normalization to glyderaldehyde-3 -phosphate dehydrogenase (GAPDH) to control for differences in starting RNA quantity. Results were expressed in terms of fold change relative to normal brain.
  • NASH National Institutes of Health
  • Figures 1A-1D show levels of upregulation of certain inflammation-linked transcripts in mouse CJD brain or microglia at early timepoints after inoculation.
  • Figures 1 A and IB show the results from brain tissue;
  • Figures 1C and ID show the results obtained from samples more than 95% pure in microglial cells. These panels also include results when poly I:C is employed.
  • Figure 2 shows the results graphically for some markers. These alterations occurred long before any PrP-res or clinical disease signs, and often persisted throughout the course of disease. L-selectin and serum amyloid A3 (SAA3) exhibited some of the earliest changes, with induction occurring as soon as 10-20 days after inoculation.
  • SAA3 serum amyloid A3
  • Flow cytometry analysis of leukocytes from CJD brain revealed no increase in cells positive for both cell-surface L-selectin and the T cell marker CD3, which demonstrates that upregulation of L-selectin mRNA reflects a response of resident glial cells to CJD infection, and is not due to transient influx of T lymphocytes positive for L-selectin.
  • the myeloid cell recruitment factors MIP-1 ⁇ , MIP-1 ⁇ , and MCPl were induced, as well as IL-l ⁇ and TNF ⁇ proinflammatory activators. Each of these was upregulated at least tenfold and then decreased somewhat prior to massive upregulation of 20-100 fold during neurodegenerative stages of disease.
  • Microglia treated with particular TLR agonists exhibit transcriptional changes that partially overlap with those observed in microglia isolated from CJD brain, and peripherally inoculated scrapie can be retarded by injection of unmethylated CpG oligonucleotides known to bind TLR9, Sethi, S., et al, Lancet (2002) 360:229-230.
  • TLR9 mRNA was detectable in spleen cells by RT-PCR, no transcripts could be detected in whole brain or adult microglia using this sensitive assay.
  • Transcripts for TLR2 and TLR3 were detected in brain, but they remained unchanged over the course of CJD infection, but TLR4 mRNA levels showed 10-fold increases at 40 days and 15-fold at 90 days.
  • Example 2 Levels of TSE Markers as a Function of Time [0042] As described in Example 1, levels of various markers were determined in the brain of the mouse CJD brain at various time points. The results are shown in Table 1 below. Table 1 Days — > ⁇ 10 20 30 40 50 60 70 80 90 100 110 120 130 SAA3 + + +++ +++ + + + + + + ++ + + ++ L-selectin + ++ ++ + + + + MlP-l ⁇ +++ ++ + ++ + +++ H-++ ++++ ImmResGl ++ + + + + + + ++ ++ -H- ++ MIP-1 ⁇ ++ + +++ +++ +++ ++++ IFI202 ++ + + + + + ++ +++ IFI204 ++ + + + + ++ + ++ CD84 ++ ++ + + + + + + + + ++ ++ ++ CD72 ++ ++ + ++ + ++ + ++ + IL-l ⁇ ++ + + + + ++ -f
  • early markers of the condition that appear prior to even 50 days subsequent to inoculation include L-selectin, SAA3, IFI202, TNF ⁇ , MIP-1 ⁇ , MIP-1 ⁇ , IFI204, ImmResGl, CD84, CD72, LY9, IL-l ⁇ , and MCPl.
  • Low levels of CXCLIO, CX3CR1, CXCL13, LY86, Cystatin F and RANTES also appear early. Detectable levels of some of these are maintained in a middle phase between 50 and 80 days.
  • Late phase markers which do not appear until subsequent to the 90 day timeframe required for appearance of the PrP-res include Clq ⁇ and CD88.
  • Example 3 Determination of Markers in Isolated Cell Types
  • Astrocytes were purified from normal and late-stage CJD-infected brain by centrifugation, MACS columns, and in vitro culture to obtain purities of astrocytes as assessed by glial fibrillary acidic protein of more than 95% purity.
  • cDNA arrays were probed with cDNA synthesized from the isolated astrocyte samples and signals with > 3 -fold change are listed in Table 2. As noted, in some cases the changes are greater than 10-fold.
  • Table 3 shows the levels of mRNA' s (as determined from cDNA arrays) in B cells where differences more than 2.5-fold were found for at least 2 timepoints at 10 days, 30 days and 50 days after inoculation. [0050] Similar results for myeloid cells are shown in Table 4.
  • AF03 i00_ 1 cytotoxic granule-associated RNA-binding protein 1 U00689 fibroblast growth factor receptor 1 X51893 fibroblast growth factor receptor 4 X59927 FMS-like tyrosine kinase 4 L07296 _ __ GENE GENBANK glial cell line derived neurotrophic factor family receptor alpha 2 AF002701

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Abstract

L'invention concerne des marqueurs de STE qui existent avant la formation d'une protéine prion pathologique détectable et permettent de détecter cette infection avant des signes cliniques. D'autres marqueurs permettent de détecter le niveau d'infection TSE et de le distinguer d'autres conditions qui présentent des symptômes cliniques similaires. Des déterminations sur des préparations cellulaires isolées sont particulièrement utiles, notamment celles issues de sources (non cervicales) périphériques.
PCT/US2005/007189 2004-03-04 2005-03-04 Marqueurs pre-symptomatiques pour des maladies associees a des encephalopathies spongiformes transmissibles (ste) WO2005091969A2 (fr)

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GB0619301A GB2427471B (en) 2004-03-04 2005-03-04 Pre-symptomatic markers for diseases associated with transmissable spongiform encephalopaties

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WO2005091969A8 WO2005091969A8 (fr) 2006-11-09

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EP2128612A2 (fr) 2006-09-05 2009-12-02 Hvidovre Hospital Surveillance immunologique sur la base de IP-10

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2128612A2 (fr) 2006-09-05 2009-12-02 Hvidovre Hospital Surveillance immunologique sur la base de IP-10
EP2228651A1 (fr) 2006-09-05 2010-09-15 Hvidovre Hospital Surveillance immunologique sur la base de IP-10
EP2261658A2 (fr) 2006-09-05 2010-12-15 Hvidovre Hospital Diagnostic des infections sur la base de IP-10

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WO2005091969A3 (fr) 2006-04-06
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WO2005091969A8 (fr) 2006-11-09
GB2427471B (en) 2009-07-22
WO2005091969B1 (fr) 2006-05-18

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