WO2010022398A1 - Diagnostic et traitement de troubles neuro-inflammatoires - Google Patents

Diagnostic et traitement de troubles neuro-inflammatoires Download PDF

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WO2010022398A1
WO2010022398A1 PCT/US2009/054794 US2009054794W WO2010022398A1 WO 2010022398 A1 WO2010022398 A1 WO 2010022398A1 US 2009054794 W US2009054794 W US 2009054794W WO 2010022398 A1 WO2010022398 A1 WO 2010022398A1
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individual
ngf
neuro
expression
cells
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Isaac Melamed
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Isaac Melamed
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/305—Attention deficit disorder; Hyperactivity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/306—Chronic fatigue syndrome

Definitions

  • the invention relates to the diagnosis and treatment of neuro-inflammatory disorders including biomarkers useful for identifying these disorders and pharmaceutical compositions useful for the treatment and prevention of these disorders.
  • ADHD Attention Deficit/Hyperactivity Disorder
  • NGF neurotrophic protein nerve growth factor
  • NGF plays a major role in the control of the allergic inflammatory response, thus serving as one of the critical proteins in the cross-talk between the nervous and immune systems and also to the endocrine system. There is also a need for better methods of identifying and diagnosing ADHD.
  • ADHD is a complex, multi-faceted illness, it is of primary importance that we understand the role that allergic response may play in the etiology of children diagnosed with chronic allergic conditions and ADHD, including the role that NGF may play in these disease states. There is also a need for better treatments of both ADHD and chronic allergic conditions in children and adolescents.
  • Autism is a developmental disorder characterized by abnormalities of social interaction, impairments in communication, and unusual forms of repetitive behavior. Similar to ADHD, and despite the increased prevalence of autism in our society (5- 8/10,000), the etiology of autism disorder is only speculative. To date, research has focused primarily on genetic, abnormal neuro-chemical, anatomical and immunological changes. While evidence exists of the presence of immunological abnormalities in children with autism, a comprehensive immunological study of this patient group has not been undertaken. Therefore, there is a need for greater understanding of the role that an immunological factors may play in the development and etiology of autism and the extent to which immunological disorders may link Autism and ADHD. There is also a need for better methods of diagnosing and treating autism. DESCRIPTION OF INVENTION
  • the invention provides methods of diagnosing and treating behavioral disorders that have a neurochemical and immunological component, including Attention Deficit/Hyperactivity Disorder (ADHD), autism, chronic fatigue syndrome, depression, anxiety and panic disorders.
  • ADHD Attention Deficit/Hyperactivity Disorder
  • these disease states are referred to as neuro-inflammatory disorders.
  • These methods and the compositions useful for the diagnosis and treatment of these disease states resulted from studies on the interaction between nervous and the immune system and the role of the nerve growth factor (NGF) receptor and its relation to CD40L signaling and the production of anti- myelin antibodies (AMA), in this interaction. Without intending to be bound by any one theory, these studies indicate that a cascade of events, initiated by various stimulants, i.e., environmental, food, stress or infection, may trigger an immune inflammatory cascade.
  • various stimulants i.e., environmental, food, stress or infection
  • Allergic disorders are also known to lead to significant cognitive distortions, especially during the peak allergy season. Interestingly, this effect has only been found in a subgroup of ADHD patients. This cascade may lead to a neuro-immune inflammation that presents clinically as one or more of the neuro-inflammatory disorders.
  • the inventors have demonstrated an improvement in ADHD scores by using drugs known to regulate allergic inflammation, e.g., the anti-histamine cetirizine.
  • drugs known to regulate allergic inflammation e.g., the anti-histamine cetirizine.
  • stimulant medication on allergic inflammation e.g., reduction in TSSC scores by methylphenidate.
  • the inventors have also shown that the synergistic effect has a better clinical effect on both the allergic and neurological components.
  • atopy may contribute to the pathogenesis of ADHD; b) nerve growth factor (NGF) may play a role in the pathogenesis of neuro-inflammatory disorders; c) neurological triggers, such as anxiety, stress or environmental factors, including food, may represent a subset of the triggers of that lead to the development of neuro-inflammatory disorders.
  • NNF nerve growth factor
  • NGF Upon mast activation and differentiation, NGF release is increased. The differentiation of human mast cell sub-types which could perpetuate human allergic reactions is dependent upon NGF.
  • NGF is increased in biologic fluids of allergy/immune - related diseases and has been evaluated lately as a TH2 cytokine with a modulator role in allergic inflammation and tissue remodeling. In patients with allergic bronchial asthma, for example, elevated levels of neurotrophins have been reported. In a time course study of bronchoalveolar lavage fluid, levels of NGF correlated with the inflammatory response. It has also been shown that NGF specifically enhances inflammation in allergic early-phase response.
  • NGF may play a role in the cross-talk between the nervous and the immune system. NGF may be released secondary to psychological stimuli, e.g., stress, anxiety and aggressive behavior. Thus, the atopic stimuli associated with the psychological stimuli may lead to the inflammatory cascade. As part of neuro-immune healing, an intact apoptosis process is required. There is increasing evidence that NGF also regulates a variety of immune-related processes of inflammation while intact apoptotic mechanisms need to be in place. NGF belongs to the super family of CD40 which plays a crucial role in the apoptosis process. Any failure in NGF signaling or regulation may lead to an abnormal apoptotic processes and, as a result, persistent inflammation. This persistent inflammation may be the cause of the neurological manifestations of neuro-inflammatory disorder.
  • the various neurological triggers initiate a cascade of events with the participation of mast cells, T regulatory cells and other immune related factors.
  • NGF and other neurotrophic factors released in response to these triggers may act as a cross link between the nervous and immune system, contributing to the persistent inflammation, either by failure of apoptosis processes or by the induction of pro-inflammatory cytokines.
  • Evidence accumulated by the inventors suggests that NGF may be involved in the failure of apoptotic mechanisms leading to persistent inflammation and neuro-inflammatory disorder.
  • Autism may also be linked to the pathways implicated by the inventor's evidence.
  • Various triggers like infection, environmental or nutritional triggers, may activate an immature immune system and lead to the development of neuro-inflammation that cannot be cleared, due to immature immune system.
  • the apoptosis failure can be due to CD40L/CD40 and NGF signaling. This failure may have a crucial effect on the apoptosis process and this may explain the persistent inflammation seen in autistic individuals.
  • This linkage between immunological/allergic and mental disorders may have a bi- directional mechanism: either allergic triggers induce a neuro-immune inflammation with a direct effect on the central nervous system (CNS), or CNS stimuli (e.g., stress, anxiety or other factors) release neuro-stimulants that impact allergic inflammation.
  • CNS central nervous system
  • CNS stimuli e.g., stress, anxiety or other factors
  • NGF release is increased.
  • the differentiation of human mast cell sub-types that perpetuate human allergic reactions is dependent upon NGF release.
  • any allergic stimulants including airborne, food or chemical stimulants, may up-regulate TH2 cytokines and as a result may release neurotrophins with the end result of neuro-inflammation.
  • the present invention provides methods of using anti-myelin antibodies (AMA) as a biomarker of neuro-immune inflammation in the neuro-inflammatory disorder states.
  • AMA anti-myelin antibodies
  • the present invention provides methods of using AMA as a novel marker of diagnosis and therapy in ADHD or other diseases with neuron-immune etiology, including the use of AMA as biochemical marker of efficacy of combined stimulant and anti-histamine drug treatment.
  • the MHC -restricted interaction between the antigen-presenting B cell and the antigen-specific responding T cell is critical for the initiation of the immune response.
  • MHC/T cell response (TcR) coupling is followed by the engagement of accessory molecules that stabilize the interaction and regulate subsequent cellular responses.
  • An important interaction is that of the B cell CD40 molecule with the T cell CD40 ligand (CD40L). Signals transuded by this and other interactions, in conjunction with the TcR- induced signals, promote the efficient activation of both cell types.
  • CD40L is a member of the TNF cytokine family that also induces TNF ⁇ , CD27L, CD30L, FasL and lymphotoxin (LT).
  • CD40L is a 33kDa type II transmembrane glycoprotein that is preferentially expressed on the surface of activated CD4 T cells.
  • CD40L contains a 22 amino acid N- terminal intracellular domain, a single transmembrane domain of 24 hydrophobic amino acids and C terminal 215 amino acid extracellular domain.
  • CD40L functions as a T cell growth factor: it stimulates proliferation and activates T cells via induction of INF ⁇ , TNF ⁇ and IL-2.
  • CD40 In addition to its association with non-receptor protein kinase, regions in the CD40 cytoplasm tail are important for binding signal transducing molecules, termed TNF receptor associated factors (TRAFs).
  • TNF receptor associated factors TNF receptor associated factors
  • Myelin is an electrically-insulating phospholipid layer that surrounds the axons of many neurons. Myelin promotes rapid and repetitive communication between neurons and it modulates the maturation and survival of axons.
  • the protein of particular interest that plays a dominant role is myelin basic protein. Re-myelination in both the peripheral and central nervous system is correlated with clinical recovery from certain neurological disorders.
  • Early vaccine studies were the first to introduce the concept that brain inflammation can be induced by an autoimmune reaction against the central nervous system born from clinical observations.
  • the CD40 ligand also referred to as gp39, TRAP and CD 154, is a tumor necrosis factor (TNF) family member glycoprotein present on the surface membrane of activated CD4+ T cells and a small subset of activated CD8+ T cells.
  • TNF tumor necrosis factor
  • the CD40L expression on activated T cells plays a pivotal role in B cell activation, proliferation and differentiation. Mutations in the CD40L gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM).
  • ICOS inducible costimulator
  • CD28 activated
  • ICOS-expression is found on T cells in germinal centers of lymphoid tissue where it interacts with counter-receptors on B cells that are undergoing the germinal center reaction.
  • ICOS plays a crucial role in T-cell dependent B-cell activation, differentiation and memory formation. This is underscored by the discovery that in certain patients with common variable immunodeficiency (CVID), ICOS is not expressed on activated T cells as a result of mutations in the ICOS gene.
  • CVID common variable immunodeficiency
  • a procedure to assay CD40L is a flow cytometric assay that uses whole blood to screen for abnormalities in the expression of CD40L and ICOS on the surface of in vitro activated CD4+ T cells. It has also been observed that both gene and surface expression of CD40L by activated T cells is depressed in a subgroup of common variable immunodeficiency while the lack of ICOS up-regulation on activated T cells would suggest the possibility of a genetic defect in the ICOS gene, underlying CVID.
  • One embodiment of the present invention relates to a method of diagnosing neuro- inflammatory disorders.
  • the method includes detecting aberrant levels of NGF, CD40L and/or AMA in tissue samples from an individual, wherein the expression of aberrant levels of these molecules, as compared to known or standard levels of these molecules from the same tissue samples from either or both of affected or non-affected individuals is indicative of the presence or emergence of a neuro-inflammatory disorder.
  • the individual is preferably a mammal, and more preferably, a human.
  • the method comprises detecting an aberrant level of NGF in a tissue sample of an individual wherein abnormal levels of NGF indicates the presence of a neuro-inflammatory disorder or a poor prognosis for an individual being treated for a neuro-inflammatory disorder.
  • the method comprises detecting an aberrant level of NGF expression in a cell sample from an individual wherein elevated NGF expression by the cells, indicates the presence of a neuro-inflammatory disorder in the individual or a poor prognosis for an individual being treated for neuro-inflammatory disorder.
  • the method comprises detecting an aberrant level of CD40L in a tissue sample of an individual wherein abnormally high levels of CD40L indicates the presence of a neuro-inflammatory disorder or a poor prognosis for an individual being treated for a neuro-inflammatory disorder.
  • the method comprises detecting an aberrant level of CD40L expression in a cell sample from an individual wherein abnormal CD40L expression by the cells, indicates the presence of a neuro- inflammatory disorder in the individual or a poor prognosis for an individual being treated for neuro-inflammatory disorder.
  • the method comprises detecting an aberrant level of AMA in a tissue sample of an individual wherein abnormally high levels of AMA indicates the presence of a neuro-inflammatory disorder or a poor prognosis for an individual being treated for a neuro-inflammatory disorder.
  • the method comprises detecting an aberrant level of AMA expression in a cell sample from an individual wherein elevated AMA expression by the cells, indicates the presence of a neuro- inflammatory disorder in the individual or a poor prognosis for an individual being treated for neuro-inflammatory disorder.
  • the step of detection can include contacting the sample with an antibody or antigen binding fragment thereof that selectively binds to one or more of NGF, AMA and/or CD40L.
  • sample is used herein in its broadest sense and includes biological samples such as fluids (e.g., blood, plasma, milk and serum), solid (e.g., stool), tissue, a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell, genomic DNA (in solution or bound to a solid support, such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), CDNA (in solution or bound to a solid support) and the like.
  • fluids e.g., blood, plasma, milk and serum
  • solid e.g., stool
  • tissue e.g., a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell
  • genomic DNA in solution or bound to a solid support, such as
  • the comparison of the measured value and the reference or control value includes calculating a fold difference between the measured value and the reference value.
  • the measured value is obtained by measuring the level of the prognostic biomarker gene expression in the sample, while in other embodiments the measured value is obtained from a third party.
  • fold difference refers to a numerical representation of the magnitude difference between a measured value and a reference value for either a prognostic biomarker or the indicative biomarker gene. Fold difference may be calculated mathematically by division of the numeric measured value with the numeric reference value.
  • a "reference value” or 'control value” can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value.
  • a reference value can be based on an individual sample value, such as for example, a value obtained from a sample from the individual diagnosed with a neuro- inflamatory disorder, but at an earlier point in time such as when determining whether a patient should continue treatment, or a value obtained from a sample from a patient other than the individual being tested, or a "normal" individual, that is an individual not diagnosed with a neuro-inflamatory disorder.
  • the reference value can be based on a large number of samples, such as from patients diagnosed with a neuro-inflamatory disorder or normal individuals or based on a pool of samples including or excluding the sample to be tested.
  • the expression levels of the identified biomarkers may be used alone or in combination with other sequences capable of determining responsiveness to treatment.
  • the biomarkers of the invention are used alone or in combination with each other or other gene sequences, such as in the format of a ratio of expression levels that can have improved predictive power over analysis based on expression of sequences corresponding to individual gene/proteins(s).
  • the biomarker of the invention may be identified via quantitative analysis of RNA expression using quantitative PCR. It can also be carried out using a Northern blot, microarray analysis, serial analysis of gene expression, nuclease protection assay, or other well known assays. Likewise, protein levels can be assessed by Western blot, immunohistochemistry, ELISA, and/or mass spectroscopy can also be used.
  • the invention also provides a predictor set comprising any one or more of the predictor biomarker genes of the invention.
  • the identified sequences e.g., polynucleotide or amino acid sequences of any one or more of the risk biomarkers disclosed herein may thus be used in the methods of the invention for predicting a particular patient's responsiveness to treatment.
  • a gene expression level can be obtained by any method and that the measurement level can be a absolute level, i.e., intensity level, a ratio, i.e., compared to a control level either of a reference gene or the gene itself, or a log ratio.
  • the pre-determined level may comprise performing the same gene expression determination in a control sample of cells and comparing the same to the sample obtained from patient diagnosed with a neuro-inflammatory disorder.
  • the control sample may be a plurality of samples obtained from a single or a plurality of patients that are not diagnosed with a neuro-inflammatory disorder (non-diseased cells) or a sample of cells from the same patient comprising cells that do not express aberrant biomarkers.
  • a control may be derived from patients with a good prognosis. Other controls are within the level of skill level of a skilled clinician.
  • differentially expressed gene refers to a gene whose expression is activated to a higher or lower level in a subject suffering from a disease, relative to its expression in a normal or control subject.
  • the terms also include genes whose expression is activated to a higher or lower level at different stages of the same disease. It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example.
  • Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically a neuro-inflammatory disease.
  • Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages.
  • Differential gene expression can, for example, be a measure of the "fold difference" between two samples.
  • “differential gene expression” may be considered to be present when there is at least an about 1.1 , or 1.2 or 1.5-fold difference between the expression of a given gene in normal and diseased subjects, or in various stages of disease development in a diseased subject.
  • Differential gene expression can also be measured using a p-value.
  • p-value a biomarker gene is identified as being differentially expressed as between a first and second population when the p-value is less than 0.1. In certain embodiments the p-value is less than O. 05, while in others it may be lower.
  • fold difference refers to a numerical representation of the magnitude difference between a measured value and a reference value for one or more of the biomarker genes of the invention. Fold difference is calculated mathematically by division of the numeric measured value with the numeric reference value.
  • Up-regulated refers to increased expression of a gene and/or its encoded polypeptide.
  • Increased expression refers to increasing (i.e., to a detectable extent) replication, transcription, and/or translation of any of the biomarker genes described herein since up-regulation of any of these processes results in concentration/amount increase of the polypeptide encoded by the gene (nucleic acid).
  • downstream-regulation or “decreased expression” as used herein, refers to decreased expression of a gene and/or its encoded polypeptide.
  • the up-regulation or down-regulation of gene expression can be directly determined by detecting an increase or decrease, respectively, in the level of mRNA for the gene, or the level of protein expression of the gene-encoded polypeptide, using any suitable means known to the art, such as nucleic acid hybridization or antibody detection methods, respectively, and in comparison to controls. In general, the variation in gene expression level is "statistically significant". Up- or down-regulation may be expressed as a fold-difference, e.g., genes or encoded proteins which demonstrate a e.g., 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, or more increase or decrease in gene expression (as measured by RNA expression or protein expression), relative to a control.
  • a fold-difference e.g., genes or encoded proteins which demonstrate a e.g., 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, or more increase or decrease in gene expression (as measured by RNA expression or protein expression),
  • a “control level” or “control sample” or “reference level” means a separate baseline level measured in a comparable control cell, which is generally disease free. It may be from the same individual or from another individual who is normal or does not present with the same disease from which the diseased or test sample is obtained.
  • a “reference value” can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value.
  • a reference value can be based on an individual sample value, such as for example, a "normal” individual, that is an individual not diagnosed with a neuro-inflammatory disease. The reference value can be based on a large number of samples, such as from patients diagnosed with a neuro-inflammatory disease or normal individuals or based on a pool of samples including or excluding the sample to be tested.
  • Gene expression profiles can also be displayed in a number of ways.
  • a common method is to arrange raw fluorescence intensities or ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data are arranged so genes that have similar expression profiles are proximal to each other. The expression ratio for each gene is visualized as a color. For example, a ratio less than one (indicating down- regulation) may appear in the blue portion of the spectrum while a ratio greater than one (indicating up -regulation) may appear as a color in the red portion of the spectrum.
  • Commercially available computer software programs are available to display such data including "GENESPRING” from Silicon Genetics, Inc. and “DISCOVERY” and "INFER” software from Partek, Inc.
  • protein levels can be measured by binding to an antibody or antibody fragment specific for the protein and measuring the amount of antibody-bound protein.
  • Antibodies can be labeled by radioactive, fluorescent or other detectable reagents to facilitate detection. Methods of detection include, without limitation, enzyme-linked immunosorbent assay (ELISA) and immunoblot techniques.
  • ELISA enzyme-linked immunosorbent assay
  • the inventive methods, compositions, articles, and kits of described and claimed in this specification include one or more biomarkers.
  • Biomarker” or “marker” is used throughout this specification refers to genes and/or gene sets and gene expression products that correspond with any gene the over- or under-expression of which is associated with a disease state. In some embodiments, it may be desirable to use the fewest number of markers sufficient to make a correct medical judgment. This prevents a delay in treatment pending further analysis as well unproductive use of time and resources.
  • gene expression can be measured by any method known in the art, including, without limitation on a microarray or gene chip, nucleic acid amplification conducted by polymerase chain reaction (PCR) such as reverse transcription polymerase chain reaction (RT-PCR), measuring or detecting a protein encoded by the gene such as by an antibody specific to the protein or by measuring a characteristic of the gene such as DNA amplification, methylation, mutation and allelic variation.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • the microarray can be for instance, a cDNA array or an oligonucleotide array. All these methods and can further contain one or more internal control reagents.
  • Preferred methods for establishing gene expression profiles include determining the amount of RNA that is produced by a gene that can code for a protein or peptide. This is accomplished by reverse transcriptase PCR (RT-PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyze it via microarray. A number of different array configurations and methods for their production are known to those of skill in the art.
  • cDNA complementary DNA
  • cRNA complementary RNA
  • articles of this invention include representations of the gene expression profiles useful for prognosticating, monitoring, and otherwise assessing diseases. These profile representations are reduced to a medium that can be automatically read by a machine such as computer readable media (magnetic, optical, and the like).
  • the articles can also include instructions for assessing the gene expression profiles in such media.
  • the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above.
  • the articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format. A graphical recordation is one such format.
  • the media are used to generate a report for a medical practitioner.
  • kits made according to the invention include formatted assays for determining the gene expression profiles. These can include all or some of the materials needed to conduct the assays such as reagents and instructions and a medium through which biomarkers are assayed.
  • the kit comprises a chip comprising protein markers for detecting CD40L and NGF.
  • the kit comprises a chip comprising nucleotide makers for detecting CD40L and NGF mRNA.
  • the kit comprises a chip comprising a ligand for detecting AMA.
  • the activity of CD40L is assayed, wherein a below normal activity is indicative or predictive of an immuno-inflamatory disease, such as ADHD, autism, or chronic fatigue syndrome.
  • the assay is a flow cytometric assay that uses whole blood to screen for abnormalities in the expression of CD40L on the surface of in vitro activated CD4+ T cells.
  • Another embodiment relates to a method of reducing the incidence of (or completely preventing) a neuro-inflammatory disease (for example, autism, chronic fatigue syndrome or ADHD) comprising, in an individual, particularly an infant or young child, by delaying the administration of vaccines if the individual is positive for biomarkers indicative or predictive of neuro-inflammatory diseases.
  • a neuro-inflammatory disease for example, autism, chronic fatigue syndrome or ADHD
  • the inventors have observed a delayed development of the immune system related to atopy.
  • the inventors contemplate that the neuro-inflammatory disease can be avoided if the individual in not subjected to the additional stress of the vaccine when the levels of certain biomarkers are high.
  • the methods comprises the step of (a) prior to vaccination of an individual, a biological sample is obtained from the individual; (b) assaying the biological sample for a biomarker predictive of autism, chronic fatigue syndrome, or ADHD; (c) postponing a vaccination for an individual whose biological samples assay positively for a biomarker predictive of autism, chronic fatigue syndrome, or ADHD; and (d) vaccinating the individual.
  • the postponing in (c) is for a period sufficient for the biomarker to correct to normal.
  • the biomarker is selected of the group comprising AMA, NGF, or CD40, wherein levels of AMA or NGF expression higher than normal are predictive of autism, chronic fatigue syndrome, or ADHD; and wherein levels of CD40 express lower than normal are predictive of autism, chronic fatigue syndrome, or ADHD.
  • the method further comprises the steps after (c), of obtaining a second sample from the individual, assaying the second biological sample for a biomarker predictive of autism, chronic fatigue syndrome, or ADHD, and further postponing a vaccination for the individual if the second biological sample assays positively for the biomarker.
  • atopic markers may be considered for determining if a delay in vaccination is desirable, including increased IgE, food allergies to milk, eggs or wheat, increased IL-5, and increased Eosinphilis.
  • the biomarker CRP may be considered to determine of dealy in vaccination is preferable.
  • immune markers may be considered for determining if a delay in vaccination is desirable, including decreased levels of perform, and decreased levels of Vitamin D metabolite.
  • the period of postponement of a vaccination is about 2 weeks, 1 month, 2 months, 3 months, 4 months, 5, months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 month, 12 months, 13 months, 14 month, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or more.
  • the period of postponement is about 2 years.
  • the individual to be vaccinated is about 2 months, 3 months, 4 months, 5, months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 month, 12 months, 13 months, 14 month, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 30 months, 36 months, 42 months, 4 years, 5 years or 6 years old or more.
  • the vaccination is postponed until the individual is at least about 36 months of age.
  • the family history of the individual is checked for a history of autism or atopy.
  • the individual is first screen for food allergies.
  • Another embodiment of the present invention relates to a method for the production of a monoclonal antibody that selectively binds to a specific protein form of NGF, AMA, and/or CD40L comprising: (a) immunizing a non-human mammal with a NGF, AMA, and/or CD40L protein such that antibody-producing cells are produced, (b) removing and immortalizing said antibody producing cells; (c) selecting and cloning the immortalized antibody producing cells producing the desired antibody; and (d) isolating the antibodies produced by the selected, cloned immortalized antibody producing cells.
  • the NGF, AMA, and/or CD40L protein is an aberrant NGF, AMA, and/or CD40L protein that has an altered activity compared to the wild type NGF, AMA, and/or CD40L protein.
  • Included in this embodiment is any antibody produced by the method, including a monoclonal antibody.
  • Yet another embodiment of the present invention relates to an isolated antibody that selectively binds to a NGF, AMA, and/or CD40L protein, wherein the antibody detects any aberrant form of the NGF, AMA, and/or CD40L protein.
  • Another embodiment of the invention includes a composition comprising any of the above-identified antibodies.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • Another embodiment of the invention includes the use of any of the above-identified antibodies in a composition for diagnosing a neuro-inflammatory disorder.
  • Yet another embodiment of the invention includes the use of any of the above-identified antibodies in a pharmaceutical formulation for treating a neuro-inflammatory disorder in an individual.
  • Another embodiment of the present invention relates to a method of treating a neuro-inflammatory disorder in an individual, comprising administering to the individual an antibody that selectively binds to the NGF, AMA, and/or CD40L protein(s) and inhibits the activity of at least one of these proteins.
  • the neuro-inflammatory disorder is ADHD or autism.
  • Yet another embodiment of the present invention relates to a method of preventing a neuro-inflammatory disorder in an individual, comprising administrating to the individual a therapeutically effective amount of an antibody that selectively binds to and inhibits the activity of a NGF, AMA, and/or CD40L protein.
  • Another embodiment of the invention relates to the use of any of the above- identified isolated NGF, AMA, and/or CD40L proteins in a pharmaceutical formulation. Another embodiment of the invention relates to the use of NGF, AMA, and/or CD40L proteins in a pharmaceutical formulation for treating a neuro-inflammatory disorder.
  • Another embodiment of the present invention relates to a method of treating or preventing a neuro-inflammatory disorder in an individual comprising administering to the individual an agent which modulates the activity or expression of NGF, AMA, and/or CD40L proteins in the individual.
  • the agent inhibits the expression of at least one of NGF, AMA, and/or CD40L proteins.
  • Another embodiment of the invention relates to a method for screening for compounds effective for the treatement of a neuro-inflammatory disorder comprising: (a) contacting a cell with a putative treatment compound; and, (b) detecting NGF, AMA, and/or CD40L proteins in the cell that may be indicative of successful modulation of NGF, AMA, and/or CD40L proteins by the putative test compound.
  • Another embodiment of the invention includes methods of treating an individual suffering or suspected of suffering from a neuro-inflammatory disorder.
  • the method includes administering to the individual a single pharmaceutical dosage form comprising an antihistamine and a neural stimulant or pharmaceutically-acceptable salts of these medications.
  • the antihistamine may be selected from ceterizine, fexofenadine, loratadine, and desloratadine.
  • the stimulant medication may be methylphenidate.
  • a “pharmaceutically acceptable salt” as used herein includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and that are not biologically or otherwise undesirable.
  • a compound of the invention may possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.
  • Examples of pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base, such salts including sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitromenzoates, hydroxybenzoates, methoxybenzoates
  • the active antihistamine and stimulant compounds are effective over a wide dosage range and are generally administered in a therapeutically-effective amount.
  • the dosage and manner of administration will be defined by the application of the antihistamine and stimulant agents and can be determined by routine methods of clinical testing to find the optimum dose. These doses are expected to be in the range of 0.001 mg/kg to 100mg/kg of active compound. It will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the antihistamine and stimulant compounds are administered in the form of pharmaceutical compositions. These compounds can be administered orally. Such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art.
  • the pharmaceutical compositions of the present invention contain, as the active ingredient, one or more of the antihistamine and stimulant compounds, in pharmaceutically acceptable formulations.
  • the active ingredient is mixed with an excipient, diluted by an excipient or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container.
  • An excipient is usually an inert substance that forms a vehicle for a drug.
  • the excipient when it serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • the compositions can be in the form of solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 30% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the pharmaceutical composition is delivered in via a transdermal patch.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, gum Arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • the compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • the principal active antihistamine and stimulant ingredients are mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, or tablets.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerator
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter.
  • compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in microencapsulated form.
  • the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the drugs in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • the dosage formulations provided by this invention may contain the antihistamine and stimulant compounds, either alone or in combination with other therapeutically active ingredients, and pharmaceutically acceptable inert excipients.
  • pharmaceutically acceptable inert excipients' includes at least one of diluents, binders, lubricants/glidants, coloring agents and release modifying polymers.
  • the dosage form may include one or more diluents such as lactose, sugar, cornstarch, modified cornstarch, mannitol, sorbitol, and/or cellulose derivatives such as wood cellulose and microcrystalline cellulose, typically in an amount within the range of from about 20% to about 80%, by weight.
  • diluents such as lactose, sugar, cornstarch, modified cornstarch, mannitol, sorbitol, and/or cellulose derivatives such as wood cellulose and microcrystalline cellulose, typically in an amount within the range of from about 20% to about 80%, by weight.
  • the dosage form may include one or more binders in an amount of up to about 60% w/w.
  • suitable binders include methyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, eudragits, ethyl cellulose, gelatin, gum arabic, polyvinyl alcohol, pullulan, carbomer, pregelatinized starch, agar, tragacanth, sodium alginate, microcrystalline cellulose and the like.
  • Suitable disintegrants include sodium starch glycolate, croscarmellose sodium, crospovidone, low substituted hydroxypropyl cellulose, and the like.
  • the concentration may vary from 0.1% to 15%, by weight, of the dosage form.
  • lubricants/glidants examples include colloidal silicon dioxide, stearic acid, magnesium stearate, calcium stearate, talc, hydrogenated castor oil, sucrose esters of fatty acid, microcrystalline wax, yellow beeswax, white beeswax, and the like.
  • concentration may vary from 0.1% to 15%, by weight, of the dosage form.
  • Release modifying polymers may be used to form extended release formulations containing the compounds of formula I.
  • the release modifying polymers may be either water-soluble polymers, or water insoluble polymers.
  • water-soluble polymers examples include polyvinylpyrrolidone, hydroxy propylcellulose, hydroxypropyl methylcellulose, vinyl acetate copolymers, polyethylene oxide, polysaccharides (such as alginate, xanthan gum, etc.), methylcellulose and mixtures thereof.
  • water-insoluble polymers include acrylates such as methacrylates, acrylic acid copolymers; cellulose derivatives such as ethylcellulose or cellulose acetate; polyethylene, and high molecular weight polyvinyl alcohols.
  • Coloring agents may be selected from the FDA approved colorants including, for example, Iron oxide, Lake of Tartrazine, Allura red, Lake of Quinoline yellow, Lake of Erythrosine.
  • a preferred embodiment of the invention is a mono-phasic pharmaceutical composition suitable for single dose administration for reducing the severity or occurrence of a neuro-inflammatory disorder consisting essentially of methylphenidate and an antihistamine selected from ceterizine, fexofenadine, loratadine, or desloratadine, and a pharmaceutically acceptable carrier.
  • Another embodiment of the invention relates to the use of any of the antihistamine and neural stimulant compositions described herein in the preparation of a medicament for the treatment of a neuro-inflammatory disorder.
  • CD40L Upregulation CD40L cell surface expression was measured in sodium heparin anti-coagulated peripheral blood. (EDTA interferes with the function of calcium ionophore and results in low- level upregulation of CD40L; data not shown). Lymphocyte activation and monoclonal antibody labeling were affected directly in whole blood.
  • Tube 1 CD8-FITC, IgG-PE, CD3- CyChrome (CD3 and CD8 are gating reagents and IgG-PE serves as the isotope control for positive/negative fluorescence discrimination);
  • Tube 2 CD8-FITC CD40L-PE (TRAP- 1) and CD3-CyChrome (percentage of CD3+CD8- T cells expressing the CD40L) and
  • Tube 3 CD3-FITC and CD69-PE (in vitro stimulation control).
  • CD69-PE which was purchased from Becton Dickinson. Cells were labeled with the mAb at room temperature in a dark environment for 20 min, then the red blood cells were lysed (FACSlyse, Becton
  • CD4 expressed on T cells thus preventing its use as a positive selection marker.
  • a negative gating strategy was adopted, i.e., CD40L was measured on CD3+, CD8- T cells (see figure 1).
  • Results were expressed as the %CD40L+ in vzYro-activated CD3+CD8- T cells.
  • An IgGl-PE isotope control monoclonal antibody was used to discriminate between positive and negative fluorescence, i.e., %CD40L+ was determined as the fraction of cells expressing fluorescence greater than the isotype control.
  • the percent positive was selected for analysis as the results were more consistent than those achieved by measuring the mean fluorescence intensity of CD40L expression; data not shown).
  • the upregulation of cell surface CD69 was used as a positive in vitro stimulation control, i.e., the upregulation of CD69 following incubation with PMA and ionomycin confirms in vitro lymphocyte activation.
  • the optimized assay was performed on heparinized peripheral blood samples obtained from 25 healthy control subjects in order to establish normal ranges for both %CD69+ and the %CD40L+ T cells.
  • T helper cells deliver a signal that activates APCs.
  • the licensed APC can than directly stimulate T-killer cells.
  • the key to this model is a specialized "professional" APC which has been identified as the dentritic cell.
  • Dentritic cells sample antigens in all body tissues and migrate to lymph nodes where they present antigens to T cells.
  • the molecules responsible for the interaction between the T-helper and dendrite cells are called CD40L and CD40.
  • Bennet shows that mice lacking T-helper cells cannot mount a killer response.
  • Schoneberger noticed that, in normal mice, it was possible to block the induction of a helper -dependent T-killer response by using antibodies to CD40 L. The addition of the stimulatory anti-CD40 antibody reversed this inhibition.
  • NGF The functional activity of NGF is mediated by two classes of receptors: p75 (a 75- kDa glycoprotein that belong to the superfamily of cytokine receptors which includes TNF receptors, Fas, CD27, CD40, and CD30) and a transmembrane tyrosine kinase of 140 kDa, TrkA, phosphorylated on tyrosine after binding to its ligand.
  • p75 a 75- kDa glycoprotein that belong to the superfamily of cytokine receptors which includes TNF receptors, Fas, CD27, CD40, and CD30
  • TrkA transmembrane tyrosine kinase of 140 kDa, TrkA
  • a central premise of the current application is that both CD40 and NGFR functionally regulate B cells but that the integration of the signal transudation pathways and phenotypic consequences are themselves different for NGFR and CD40, even though they are in the TNF receptor family. Further, NGFR and CD40 interaction with other growth factor signaling pathways, such as those regulated by IL-2 and IL-4, interfere differently when B cell function is examined. Thus, the regulatory roles of NGF and CD40 are often opposing in the control of B-cell function.
  • NGF and CD40 are hypothesized to be involved in neuro-immune mechanisms.
  • CD40L Upregulation Clinical Procedure: CD40L cell surface expression was measured in sodium heparin anti-coagulated peripheral blood. (EDTA interferes with the function of calcium ionophore and results in low-level upregulation of CD40L; data not shown). Lymphocyte activation and monoclonal antibody labeling were affected directly in whole blood. Briefly, 200 ul of whole blood obtained from each patient and control was added to each of two tubes and then diluted 1 :5 with RPMI 1640 (unstimulated control) or with RPMI containing phorbol myristate acetate (PMA, 15ng/ml final, Sigma, St.
  • PMA phorbol myristate acetate
  • Tube 1 CD8-FITC, IgG-PE, CD3-CyChrome (CD3 and CD8 are gating reagents and IgG-PE serves as the isotope control for positive/negative fluorescence discrimination);
  • Tube 2 CD8-FITC CD40L-PE (TRAP-I) and CD3-CyChrome (percentage of CD3+CD8- T cells expressing the CD40L) and
  • Tube 3 CD3-FITC and CD69-PE (in vitro stimulation control).
  • CD69-PE which was purchased from Becton Dickinson.
  • Cells were labeled with the mAb at room temperature in a dark environment for 20 min, then the red blood cells were lysed (FACSlyse, Becton Dickinson). The samples were washed, re-suspended in 1% paraformaldehyde and stored at 4°C until analyzed by flow cytometry as described below.
  • CD40L was measured on all CD3+ T cells i.e. both T helper (CD4+) and T suppressor/cytotoxic cells (CD8+).
  • CD4+ T helper
  • CD8+ T suppressor/cytotoxic cells
  • the CD40 ligand was measured only on activated CD4 positive T cells (only a small percentage of activated CD8 positive T cells express CD40L).
  • concentrations of PMA and ionomycin used in the stimulation protocol significantly reduced the level of CD4 expressed on T cells, thus preventing its use as a positive selection marker.
  • CD40L was measured on CD3+, CD8- T cells (see figure 1).
  • Results were expressed as the %CD40L+ in vzYro-activated CD3+CD8- T cells.
  • An IgGl-PE isotope control monoclonal antibody was used to discriminate between positive and negative fluorescence, i.e., %CD40L+ was determined as the fraction of cells expressing fluorescence greater than the isotype control. (The percent positive was selected for analysis as the results were more consistent than those achieved by measuring the mean fluorescence intensity of CD40L expression; data not shown).
  • the upregulation of cell surface CD69 was used as a positive in vitro stimulation control, i.e., the upregulation of CD69 following incubation with PMA and ionomycin confirms in vitro lymphocyte activation.
  • the optimized assay was performed on heparinized peripheral blood samples obtained from 25 healthy control subjects in order to establish normal ranges for both %CD69+ and the %CD40L+ T cells.
  • naive CD4 T cells Since it has been reported that the expression of CD40L is reduced on naive CD4 T cells as compared to memory T cells, it is possible that the naive subset is over- represented in autistic children.
  • the monoclonal antibody combination will be CD4, CD45RA and CD62L. Becton Dickenson/Pharmingen will provide these antibodies.
  • the inventors also contemplate measuring the subset composition of the peripheral blood (CD3, CD4,CD8, CD20, NK cells ,106 ,5C8 and anti-TRAP; again, all the antibodies will be provided by Becton Dickinson /Pharmingen ).
  • the inventors analyzed the CD40L expression in 39 children with autism.
  • the inventors divided the autistic group to two subgroups; (1) autistic children with recurrent infections and (2) autistic children with no recurrent infections .
  • CD40L in activated T cells obtained from autistic children with recurrent infections was significantly lower than that of autistic children with no infections or from normal healthy controls.
  • Anti-Myolin Antibodies as Diagnostic Marker We followed 20 children, ages 8 - 17 years old with co-morbid diagnosis of atopy and ADHD. In addition to attention and behavioral measurements, levels of AMA were tested pre- and post-therapy.
  • Atopy was defined as having a clinical diagnosis of allergic rhinitis, eczema or asthma.
  • An associated clinical diagnosis with positive skin tests or RAST tests to prevalent allergens including food was also necessary to participate, (prick wheal >3mm over the negative control; intradermal wheal >5mm over the negative control).
  • Diagnosis of ADHD was based on Connor's evaluation (4, 5). Connors' Rating
  • Scale is based on a 27-item questionnaire, which evaluates attention and other problem behaviors in children and adolescents, was utilized by parents, teachers or alternate caregivers.
  • the AMA assay was performed by Quest, LabCorp. AMA levels were elevated in 16 of the 20 patients pre-treatment: 1) IgM subtype above 1 :32 in 4 subjects; 2) IgG subtypes in 12 patients above normal levels. After 1 month of therapy, the 12 patients with increased levels of AMA IgG subtypes reversed from positive levels to negative while in the 4 patients with IgM subtype levels remained positive. In addition, the attention and atopic scores of the 12 patients correlated with the normalization in AMA IgG levels, i.e., the attention and atopic scores showed an improvement while the AMA IgG levels decreased. CD45RA + and CD45RO+ in autistic children
  • Human T lymphocytes can be phenotypically divided into two major subsets, based on the expression of different isoforms of the CD45 surface Ag. These subsets, CD45 RA+ and CD45RO+, differ in their ability to synthesize different lymphokines and in their ability to respond to recalling Ags.
  • CD40L is expressed in higher levels on CD45RA+ T cells as compared to CD45RO+T cells following CD3 -stimulation. (Fig)
  • the in vitro conversion of CD45RA+ T cells into CD45RO phenotype is accompanied by a loss in the ability to express the CD40L.
  • T cell isoforms CD45RA+ and CD45RO+ T cells from autistic children and controls were stimulated in flat bottom, 24-well plates for the indicated period of time. Cells were collected in 12x75 mm plastic centrifuge tubes and T cells were stained for CD40L expression using Ab 39-1.106 to human CD40 ligand (lug/ml) followed by FITC- conjugated sheep anti-mouse AB (Sigma).
  • RNAse protection assay total cellular RNA will be isolated from T cells of normal children and autistic children. The cells are stimulated with various combinations of stimulants. Twenty micrograms of total RNA from T cells were hybridized with CD40 and GAPDH riboprobes at 42° C overnight in 20 ⁇ l of 4OmMPIPES pH 6.4 ,80% deionized formamide, 40OmM sodium acetate and ImM EDTA. The hybridized mixture are then treated with RNAse A/Tl at room temperature for 1 h, analyzed by 5% denaturing (8M urea) polyacrylamide gel electrophoresis, and the gels exposed to X-ray films.
  • the protected fragments of the CD40 and GAPDH ribprobes are 419 and 212 nucleotide in length respectively. Quantitation of the protected RNA fragments are performed by scanning with a Phosphoimager (molecular dynamics). Values for CD40 mRNA expression are normalized to GAPDH mRNA levels for each experimental condition. GAPDH mRNA is utilized as a "housekeeping gene", as its levels are not affected by cytokine treatment. In order to determine whether or not CD40L genes are mutated in autism, the inventors contemplate setting up T cell lines from autistic children. The T cell lines will be IL-2 dependent cells that can be cloned into CD4+ cells. Regulation ofCD40L The role ofCD28
  • CD40L The expression of the CD40L on T cells is tightly regulated, presumably, to minimize bystander B cell activation.
  • the protein is rapidly induced following the stimulation of T cells via TcR-CD3 complex and than wanes within some 24 hours.
  • the interaction of CD40L-bearing T cells with CD40+ cells induces very rapid down regulation of the protein. This is initially due to receptor-mediated endocytosis but is hypothesized to involve downregulation of mRNA for the CD40L, as well.
  • CD40L expression seems to be controlled by similar mechanisms to those which regulate production of many T cell derived cytokines. Hence, the immunosuppressive drug cyclosporine and FK506 abrogate the expression of the CD40L.
  • T cells obtained from autistic children and controls are cultured (10x106) in supplemented RPMI 1640 medium plus 5% FCS in flasks coated with anti-CD3 (lOug/ml, for 24 h at 4 co) in the presence or absence of anti-CD28.
  • CD40L expression are measured at different time frames to obtain the kinetic of up and down regulation of CD40L. Functions ofCD40 in vitro
  • CD40 acts in concert with either cytokines or other receptor- ligand interactions.
  • cytokines For example, for isotope switching, the process is initiated by CD40 whereas the specificity of the isotope is determined by different cytokines. In humans, this concerns IL-4/IL-13 and IL-IO.
  • CD40 antigen-presenting cells like monocytes and dendritic cells
  • CD40 ligation on monocytes and dendritic cells results in enhanced survival of these cells as well as in the secretion of cytokines, such as IL-I, IL-6, IL-8, IL-IO, IL-12 and TNF- ⁇ .
  • cytokines such as IL-I, IL-6, IL-8, IL-IO, IL-12 and TNF- ⁇ .
  • CD-40 ligation considerably alters these APC phenotypes by up-regulating the expression of the co-stimulatory molecules such as ICAM-I, LFA-3.
  • the interaction between CD40 and CD40L therefore, has important consequences for both APC and T cell function. Interrupting CD40/CD40L interactions during T-cell dendritic cell cocultures results in reduced T-cell proliferation, possibly as a consequence of both altered CD40 signaling to the APC and altered CD40L signaling to the T cells.
  • CD40L expression seems to be controlled by mechanisms similar to those which regulate the production of many T-cell derived cytokines.
  • the inventors contemplate delineating the role of CD40L expression in regulation of different cytokines in autistic and normal children.
  • the inventors contemplate determining the effects of the addition of anti-CD40 antibody on IL-12, INF ⁇ and IL-6 in autistic and normal children and correlate these findings with the levels of expression of CD40L on circulating T cells. INF ⁇
  • CD40L /CD40 stimulation in INF ⁇ production can occur through at least two mechanisms. The first, through direct induction of IL-12 from APC, such as dentritic cells, and the second, through the ability of CD40 /CD40 ligation to enhance the expression of cell surface molecules, such as Bl, thus increasing T cell stimulation and production of INF ⁇ . Moreover, in CD40L knock out mice, the impairment of THl type immune responses was shown to be the result of the inability of macrophages to produce IL-12 and low levels of INF ⁇ .
  • PMBC cells are incubated in a total volume of 1 ml in 24-well plates. Cells are stimulated with anti-CD40 mAb (250LVmI). Supernatants are harvested after 5 days cultures. INF ⁇ accumulation in the supernatants is determined using an ELISA kit obtained from R&D systems.
  • IL-6 IL-6 is also involved in the regulation of CD40L. IL-6 is expressed by a number of cell types and induces a wide variety of responses, including differentiation of B cells and augmentation of T cell response. The expression of IL-6 is regulated primarily through the binding of NFkB transcription factor to IL-6 promoter sequences.
  • IL-6 can be an intermediary in sequential signaling pathways activated through CD40.
  • IL-6 To study the expression of IL-6, PMBC cells are incubated in a total volume of 1 ml in 24-well plates. Cells are stimulated with anti-CD40 mAb (250LVmI). Supernatants are harvested after 5days cultures. IL-6 accumulation in the supernatants is determined using an ELISA kit obtained from R&D systems. IL- 12
  • IL- 12 has been shown to play a critical role in the regulation of immune responses in various autoimmune disease models.
  • IL- 12 is important in influencing the differentiation of na ⁇ ve CD4 T cells toward an interferon- ⁇ -producing ThI- type cell, as well as enhancing cytotoxic T-cell mediated lysis and natural killer cell activity.
  • ThI responses play an important role in mediating protective immunity against certain intracellular infections as well as in initiating or exacerbating specific autoimmune response. Renoux and his colleagues hypothesized that impaired T-cell function in autistic children reflected an imbalanced control of T cell function by the left and right hemisphere.
  • IL- 12 production was not effected after lesion to the right cortex.
  • CD40/CD40L costimulatory pathway has been demonstrated to be one of the major regulators of IL- 12 induction, the inventors contemplate a role for IL- 12 after exogenous CD40L/CD40 stimulation in autistic children.
  • PBMCs from autistic and healthy controls are isolated by lymphocyte seperation.
  • PMBC is stimulated with PHA, in the presence and absence of CD28 Ab.
  • Supernatants are collected after 40 hours to measure the production of IL-12.
  • An IL-12 specific ELISA that detects P70 heterodimer is used (R&D Systems; lower limit of detection, 7.8pg/ml. Delineating the role ofCD40L in T and B cell signaling in autistic children
  • CD40 signal transudation has resulted in a more precise picture of the different mediator pathways involved in this process.
  • CD40 has no kinase domain and no known consensus sequence for binding to kinases
  • CD40 ligation activates several second messenger systems, including protein tyrosine kinases, PI-3 kinase and phospholipase C ⁇ 2, as well as the serine threonine stress activated protein kinases.
  • These different activation pathways result in the activation of various transcription factors, including NFkB, c-jun and NF-AT.
  • the inventors have demonstrated that CD40 stimulation of human B cells leads to selective activation of the c-jun kinase /stress activated protein kinase pathway.
  • TRAF TNF receptor associated factor
  • TRAF3 is a 62 KD intracellular protein which is expressed in almost all cell types. TRAF3 has also been demonstrated to associate with EBV, thus indicating that EBV uses CD40 signaling. CD40 also associates with TRAF2, a molecule, which mediates NFkB activation following either CD40 or TNF stimulation.
  • Antigen-receptor mediated activation of normal human B and T cells is associated with early intracellular metabolic changes, including phosphoinositide turnover, tyrosine phosphorylation and cytoskeletal reorganization. These different pathways operate in concert to transmit signals which, ultimately, trigger cell growth, differentiation and death.
  • the inventors have shown that ligand-induced PTK activity precedes phosphoinositide hydrolysis and actin reorganization in the signal transduction cascade.
  • the inventors have also demonstrated that antigen-induced tyrosine phosphorylation is essential for the activation of these secondary pathways differentiation of B and T cells and that they are key instruments in regulating phosphatidylinositol metabolism and cytoskeletal reorganization.
  • Ligation of CD40 initiates a distinct series of events which result in no increase in the cytosolic Ca concentration and inconsistent results regarding Src kinase activation.
  • NF-kB is a pleiotropic transcription factor that is involved in the regulation and expression of No genes.
  • IL-4 and anti-CD40 induced a strong activation of NFkB.
  • NF -KB transcription activity is modulated by CD-40 and provides a signal required for B-cell activation and differentiation, including B-cell immunoglobulin class-switching.
  • the Epstein Barr virus (EBV) can immortalize primary B-lymphocytes.
  • EBV immortalization is an induction of CD40 independent NF -KB transcription activity mediated by binding between the EBV encoded LMP-I gene product and TRAF (TNF ⁇ receptor activating factor).
  • TRAF TRAF
  • the interaction between LMP-I and TRAF is thought to account for the observation that EBV immortalized B-lymphocytes undergo CD40 independent class switching since the CD40 signal is in part mediated through NF -KB activation.
  • EBV immortalized B cell lines will be generated from patients with autism and from controls. EBV immortalized B-cell lines generated from patients with autism and controls will be used for two purposes: 1) to determine NF -KB transcription activity of patients Vs controls and 2) to characterize CD40 gene structure in-patients and controls. NF -KB transcription activity will be determined by Electrophoresis Mobility Shift Assay (EMSA) of immortalized B-lymphoblastoid cell lines using a rapid protocol for nuclear protein extraction (2) and an oligonucleotide derived from the human high affinity NF -KB binding sequence (3).
  • ESA Electrophoresis Mobility Shift Assay
  • EMSA analysis of immortalized B-lymphoblastoid cell lines demonstrates that levels of NF- ⁇ B binding proteins are remarkably constant in independently derived cell lines from a single patient ( Figure). Basal levels of NF- ⁇ B binding proteins in immortalized cell lines and controls will be determined by EMSA to establish whether the defect in CD40 activation in patients can be bypassed by EBV-mediated TRAF activation or whether these patients have an intrinsic defect in activation that cannot be bypassed by TRAF activation.
  • EBV immortalized B-cell lines generated from patients with autism and controls will also provide a stable source of DNA to examine CD40 gene structure and to determine whether the defects in CD40 in-patients result from altered gene expression or protein sequence. Once cell lines are established, they can be stored indefinitely and used in additional studies.
  • Nerve growth factor is a well-characterized neurotrophic protein essential for the survival and differentiation of sympathetic and sensory neurons in the peripheral nervous system and for cholinergic neurons in the central nervous system.
  • the effect of NGF is initiated through the formation of a receptor-ligand complex followed by its translocation from the cell surface to the cytoplasm.
  • NGF is involved in inflammatory responses.
  • NGF has been found to increase the number of mast cells in neonatal rats, to enhance both in vivo and in vitro chemotaxis for human polymorphonuclear neutrophils and to induce hyperplasia of mast cells.
  • NGF receptors have been demonstrated on rat and human lymphocytes.
  • NGF has been shown to enhance lymphocyte proliferation of both B- and T- cell populations and to stimulate production of IgG4.
  • NGF shares structural homology with the B cell antigen, CD40.
  • NGFR and CD40 have been suggested to be members of a newly recognized family of cysteine- rich cytokine receptors which play a role in B cell activation.
  • anti-CD40 and NGF appeared to exert opposite effects on the production of IgE by IL-4.
  • Recent studies in our lab showed that NGF could antagonize the effect of IL-4 on IgE secretion while anti-CD40 could not.
  • NGF represents a possible adapter between the immune and nervous system and may modulate the B cell function either directly or by interacting with other cytokines.
  • studying the role of NGF in regulation of the signaling events in conjunction with CD40 may provide further understanding in the control of neuro- immune.
  • NGF is an adapter with a functional link between the nervous and the immune systems and that its interaction with different cytokines and molecules leads to a specific signaling cascade. This cascade may regulate by various cellular components, eventually leading to determination of the decision-making point for the B-lymphocytes to participate in neuro-immune inflammation.
  • NGF-tyrosine kinase(s) determines the specificity of the biological responses of various cells involved in immune-regulation. Abnormal expression of such growth factor receptor-linked result in neuro-immune inflammation. NGF induces B cell proliferation and leads to progression signals.
  • NGF can induce the proliferation of B- lymphocytes and provide progression signals to competent B-lymphocytes.
  • the inventors studied the signaling cascade in B- lymphocytes activated by NGF in normal and autistic children. NGF induces a rapid increase in tyrosine phosphorylation in human B- lymphocytes
  • gpl40trk contains an intrinsic tyrosine kinase activity, which is activated by NGF binding.
  • NGF tyrosine kinase activity
  • the inventors used western blotting with anti-phosphotyrosine antibodies to test whether NGF would increase tyrosine phosphorylation in human B lymphocytes. The inventors have consistently observed that NGF induced a time and concentration dependent increase in tyrosine phosphorylation of four bands with molecular masses of 140, 110, 85 and 42 kDa.
  • Tyrosine phosphorylation was detected within 1 min of addition of NGF. Phosphorylation detected on these western blotting analyses were strictly the result of tyrosine residues because the anti-phosphotyrosine antibodies used have been demonstrated to react specifically with phosphotyrosine and not with phosphoserine or phosphothreonine.
  • the inventors contemplate a method of studing the phosphorylation events occuring in B lymphocytes isolated from autistic children.
  • Gpl40trk mediates the signaling cascade induced by NGF It has been previously shown that the addition of NGF to PC 12 cells results in the rapid phosphorylation of gpl40trk receptors, followed by the induction of early gene expression.
  • the inventors examined the levels of tyrosine phosphorylation of gpl40/? ⁇ in B lymphocytes treated with NGF. In B cells, the inventors found a rapid increase in phosphorylation level of gpl40/?a receptor. NGF induces tyrosine phosphorylation on PLC- ⁇ l.
  • PLC protein-tyrosine kinase activity
  • PLC functions as the amplifier of the classical Ptdlns pathway.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • PLC- ⁇ l is the PLC isozyme that mediates PDGF- induced inositol phospholipid hydrolysis and that phosphorylation on tyr-783 and tyr-1254 are essential for PLC- ⁇ l activation.
  • PLC- ⁇ l is a substrate of NGF receptor activated tyrosine kinase. NGF induces inositol phosphate production.
  • NGF induces tyrosine phosphorylation on proteins with estimated sizes of 140 and 85 kDa
  • one of the other PLC isozymes such as PLC- ⁇ l or PLC- ⁇ l
  • PLC- ⁇ l PLC- ⁇ l
  • PLC- ⁇ l PLC- ⁇ l
  • NGF receptor mediates the activation of the Ptdlns pathway using a PLC isozyme other than PLC- ⁇ l, it should hydrolyze phosphatidylinositol-4, 5-bisphosphate, thus generating inositol phosphates.
  • the inventors contemplate a method of measuring the products of hydrolysis of inositol trisphosphates. NGF mediates changes in cytosolic free Ca2+.
  • [Ca2+]I One of the earliest intracellular events associated with binding of ligands to the antigen receptors on B cell receptors is the rapid increase in [Ca2+]I. This increase in [Ca2+]I is considered essential for activation of specific protein kinases associated with these receptor systems.
  • the rise in [Ca2+]I is derived from both an InsP3 -sensitive intracellular pool and from transmembrane influx of Ca2+. Since NGF may induce an increase in InsP3 concentration it may also provide a [Ca2+]I transient by increasing transmembrane influx.
  • NGF leads to activation of phosphatidylinositol-3 Kinase
  • tyrosine kinase receptors may be their differing interaction with effector proteins containing src homology domains (SH2).
  • SH2 effector proteins containing src homology domains
  • the inventors have determined that, in B cells, NGF leads to phosphorylation of PI-3kinase.
  • the inventors contemplate a method of determining the PI-3 kinase activity of B cells stimulated for different periods of time with NGF.
  • This enzyme phosphoeylates PI on the D3 position of the inositol ring, leading to the formation of the novel phosphoinositides.
  • the stimulation of PI-3kinase activity represents another early cellular response that NGF shares with a number of growth factors and mitogens. These factors may produce similar, but not identical, early signaling events.
  • the inventors contemplate a regulation pathways by which different cytokines (IL-1)
  • the RAS proto-oncogenes encode small plasma membrane associated proteins that bind quanine nucleotides and possess intrinsic GTPase activity, alternating between active GTP-bound and inactive GDP-bound forms.
  • RAS proteins have been modified by the production of RAS proteins to bind quanine nucleotides and possess intrinsic GTPase activity, alternating between active GTP-bound and inactive GDP-bound forms.
  • NGF nerve growth factor
  • anti-IgM antibody leads to the early tyrosine phosphorylation of p95vav, which is expressed exclusively in hematopoietic cells; NGF, similar to crosslinking the BCR, also results in the rapid activation of Ras.
  • the phosphorylation of Vav and activation of Ras triggered by NGF is mediated through Trk tyrosine kinase, whereas signaling through the BCR uses a different tyrosine kinase.
  • the inventors also showed that NGF and anti-IgM induces tyrosine phosphorylation of She and demonstrated its association with Grb2. Vav and Ras with some adaptor proteins like She and Grb2 appear to serve as a link between different receptor-mediated signaling pathways and, in human B cells, may play an important regulatory role in neuro-immune interactions.
  • the inventors contemplate verve/RAS experiments to further characterize the signaling pathways that mediate Vav and Ras activation and delineate the role of CD40 in the signaling cascade, specifically in lymphocytes from normal and autistic children and in B cells, and to delineate the role of other cytokines (IL-4, IL-2) in RAS activation in NGF stimulated B cells.
  • IL-4, IL-2 cytokines

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Abstract

L'invention concerne des procédés de diagnostic d'un trouble neuro-inflammatoire en analysant les niveaux d'expression de biomarqueurs, y compris NGF, CD40L et AMA, dans l'échantillon biologique provenant d'un individu, l'expression de niveaux aberrants des biomarqueurs, par comparaison à des niveaux d'expression connus ou standard du ou des biomarqueurs des échantillons biologiques identiques provenant d'individus non affectés, indiquant la présence ou l'émergence d'un trouble neuro-inflammatoire.
PCT/US2009/054794 2008-08-22 2009-08-24 Diagnostic et traitement de troubles neuro-inflammatoires WO2010022398A1 (fr)

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US6225067B1 (en) * 1999-04-13 2001-05-01 BIOMéRIEUX, INC. Methods and devices for performing analysis of a nucleic acid sample
US20080182978A1 (en) * 2002-12-24 2008-07-31 Rinat Neuroscience Corp Anti-ngf antibodies and methods using same
US20050192290A1 (en) * 2004-01-13 2005-09-01 Isaac Melamed Treatment of behavioral disorders
US20060051345A1 (en) * 2004-06-04 2006-03-09 Genentech, Inc. Method for treating multiple sclerosis
US20070254314A1 (en) * 2004-09-16 2007-11-01 Geier Mark R Methods of treating autism and autism spectrum disorders
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