EP1423144A2 - Methods and compounds for disruption of cd40r/cd40l signaling in the treatment of alzheimer's disease - Google Patents
Methods and compounds for disruption of cd40r/cd40l signaling in the treatment of alzheimer's diseaseInfo
- Publication number
- EP1423144A2 EP1423144A2 EP02753530A EP02753530A EP1423144A2 EP 1423144 A2 EP1423144 A2 EP 1423144A2 EP 02753530 A EP02753530 A EP 02753530A EP 02753530 A EP02753530 A EP 02753530A EP 1423144 A2 EP1423144 A2 EP 1423144A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cd40l
- cd40r
- amyloid
- app
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5058—Neurological cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4709—Amyloid plaque core protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/525—Tumor necrosis factor [TNF]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
-
- 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
- G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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- 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
Definitions
- a ⁇ ⁇ -amyloid
- AD Alzheimer's disease
- Tg APP S amyloid precursor protein
- AD Alzheimer's disease
- a ⁇ ⁇ -amyloid peptide
- APP amyloid precursor protein
- transgenic mice that overexpress the "Swedish” APP mutation Tg APP SW
- NSAID ibuprofen
- these animals show reduction in A ⁇ deposits, astrocytosis, and dystrophic neurites correlating with decreased microglial activation (Lim et al, "ibuprofen suppresses plaque pathology and inflammation in a transgenic mouse model for Alzheimer's disease,” J Neurosci. (2000) 20:5709-14).
- bigenic mice that overexpress human APP and transforming growth factor ⁇ l also demonstrate reduced parenchymal A ⁇ deposition associated with an increase in microglia positive for the F4/80 antigen (Wyss-Coray et al, "TGF-betal promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice,” Nat. Med. (2001) 7:612-18).
- CD40 is a ⁇ 45 kDa key immunoregulatory molecule, which plays a critical role in immune cell activation.
- ligation of B cell CD40R promotes B cell proliferation after antigenic challenge, resulting in differentiation into antibody-secreting plasma cells.
- Blockade of the CD40R-CD40 ligand (CD40L) interaction in vivo inhibits activated T cell-dependent interleukin-12 secretion by antigen presenting cells (Grewal et al, "Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis," Science (1996) 273:1864-7; Stuber et al, "Blocking the CD40L- CD40 interaction in vivo specifically prevents the priming of T helper 1 cells through the inhibition of interleukin 12 secretion," /. Exp. Med. (1996) 183:693-8).
- CD40 is expressed on cultured microglia at low levels, and CD40R expression is markedly enhanced on these cells by the pro- inflammatory cytokine interferon- ⁇ as well as A ⁇ (Carson et al, "Mature microglia resemble immature antigen-presenting cells,” Glia (1998) 22:72-85; Tan etal, "Activation of microglial cells by the CD40 pathway: relevance to multiple sclerosis,” J. Neuroimmunol (1999) 97:77-85; Tan etal, "Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation,” Science (1999) 286:2352-55).
- a ⁇ and CD40L synergistically stimulate microglia to secrete TNF- ⁇ , resulting in induction of neuronal injury in vitro, effects that are not observed in the presence of low levels of A ⁇ alone (Tan et al, "Microglial activation resulting from CD40R-CD40L interaction after beta-amyloid stimulation,” Science (1999) 286:2352-55).
- CD40L and its receptor CD40R
- CD40R CD40L and its receptor
- AD brain There is mounting evidence that products of the inflammatory process in AD brain exacerbate AD pathology. Many of these inflammatory proteins and acute phase reactants such as alpha- 1-antichymotrypsin, transforming growth factor ⁇ , apolipoprotein E and complement factors are produced by activated glia, are localized to A ⁇ plaques, and have been shown to promote A ⁇ plaque “condensation” or maturation (Nilsson et al, "Alpha- 1- antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease," J. Neurosci.
- the subject invention provides methods of treating neuronal inflammation, brain injury, tauopathies, or an amyloidogenic diseases, comprising the administration of therapeutically effective amounts of a composition comprising a carrier and an agent that interferes with the interaction of CD40L and CD40R to an individual afflicted with an amyloidogenic disease. Also provided are methods and/or assay systems for the identification of compounds or other small molecules capable of disrupting the CD40R/CD40L signaling pathway.
- the subject invention provides a method of testing a compound suspected of modulating the CD40L/CD40R signaling pathway by interfering with CD40L/CD40R signaling pathway comprising: contacting a first sample of cells with CD40 ligand and measuring an inflammatory response; contacting a second sample of cells with a compound and CD40 ligand, and measuring an inflammatory response; comparing said inflammatory response of said first sample of cells with said inflammatory response of said second sample of cells.
- compounds modulate the CD40L/CD40R signaling pathway by interfering with the association of CD40L and CD40R, by interfering with components of the signaling pathway upstream or downstream of the CD40L/CD40R interaction, or by interfering with the trimerization of CD40R.
- compounds or small molecules that interfere with TNF receptor-associated factors (TRAFs) are contemplated.
- the cell samples are obtained from, or derived from, the central nervous system (CNS; e.g., biopsied materials obtained from humans), animal models, or peripheral sources.
- the animal model cell samples comprise intact animals art recognized as models for Alzheimer's Disease or for the study of the CD40L/CD40R signaling pathway.
- the animal models may be transgenic or non- transgenic and non-limiting examples of these models include mice, worms, or flies; cells obtained from these animal models can be immortalized and cultured as cell lines.
- Cell samples can also include immortalized and non-immortalized cell lines derived from, for example, human, higher primate, primate, murine sources.
- the subject invention also provides a method for testing a compound suspected of modulating the CD40L/CD40R signaling pathway by interfering with CD40L/CD40R signaling pathway comprising, said method comprising: a. contacting CNS cells with CD40 ligand and said compound and measuring an inflammatory response; b. contacting peripheral cells with CD40 ligand and said compound and measuring an inflammatory response; c. contacting CNS cells with a stimulator of the CD40 pathway and a compound and measuring an inflammatory response; d. contacting peripheral cells with a stimulator of the CD40 and said compound and measuring an inflammatory response; e. contacting CNS cells with an inhibitor of the CD40 pathway and said compound and measuring inflammatory response; f. contacting peripheral cells with an inhibitor of the CD40 pathway and said compound and measuring inflammatory response; and g. comparing said inflammatory responses, whereby the CD40-modulating activity of said compound is tested.
- these methods measure the levels of various markers, or combinations of markers, associated with the inflammatory response by measuring the levels of one or more markers.
- Cytokine markers can be selected from the group consisting of tumor necrosis factor, interleukin 1, interleukin 6, interleukin 12, interleukin 18, macrophage inflammatory protein, macrophage chemoattractant protein, granulocyte- macrophage colony stimulating factor, macrophage colony stimulating factor and various combinations of these cytokines.
- the methods measure levels or amounts of one or more markers selected from the group consisting of glutamate release, nitric oxide production, nitric oxide synthase, superoxide, superoxide dismutase and various combinations of these markers.
- the methods set forth herein can also measure a major histocompatibility complex molecule, CD45, CD l ib, integrins, or a cell surface molecule as a marker of the inflammatory response. Yet other embodiments measure levels, amounts, or deposition of proteins on cells wherein said proteins are selected from the group consisting of A ⁇ , ⁇ -amyloid precursor protein, a fragment of a ⁇ -amyloid precursor protein, and combinations of these proteins.
- Stimulators and inhibitors according to the subject invention can be agonistic or antagonistic antibodies.
- the subject invention also provides a method for testing a compound for its ability to modulate CD40L/CD40R interactions comprising contacting a CD40 receptor and a CD40 ligand with said compound and measuring the binding of said CD40 receptor with said CD40 ligand.
- compounds can bind to CD40L or CD40R.
- the compounds can be small molecules or antibodies specific for CD40L or CD40R.
- the subject invention also provides methods of conducting in vivo assays for compounds that are capable of modulating the CD40/CD40R signaling pathway comprising administering to an animal model, or a human, an agent or compound that modulates the signaling pathway, and measuring an the animal's responsiveness to the compound.
- the method can be practiced with agents as described supra or soluble CD40L, an antibody against CD40 that inhibits the CD40 pathway, an antibody against CD40 ligand that inhibits the CD40 pathway, an antibody against CD40 that stimulates the CD40 pathway, an antibody against CD40 ligand that stimulates the CD40 interaction with CD40 ligand, a compound that blocks the CD40 pathway, a compound that interrupts the CD40 interaction with CD40 ligand, a compound that stimulates the CD40 pathway, or a compound that stimulates the CD40 interaction with CD40 ligand.
- Animals can be examined for improvements in conditions described supra or for improvements in ⁇ -amyloid deposition, soluble ⁇ -amyloid, inflammatory markers, microglial activation, astrocytic activation, neuronal apoptosis, neuronal necrosis, brain injury, tau phosphorylation, or tau paired helical filaments.
- non-human transgenic animal model comprising one or more of the following: transgenic amyloid-precursor protein, overexpressed transgenic presenilin protein, overexpressed transgenic CD40 receptor, overexpressed transgenic CD40 ligand, and/or tau protein or mutants of the tau protein.
- FIG. la-ln Microgliosis and astrocytosis are reduced in TgAPP/CD40L def. mice by 16 months of age. Panels are representative lOx bright-field photomicrographs. a-f, mouse brain sections stained with anti-CD l ib antibody; left column represents sections from TgAPP sw mice, and sections shown on the right were taken from TgAPP sw /CD40L def. mice.
- Panels a and d represent cingulate cortices (CC); b and e, hippocampi (H); and c and enthorinal cortices (EC), g-l, mouse brain sections stained with anti-GFAP antibody; left column represents sections from TgAPP sw mice, and sections shown on the right were taken from Tg APP SW /CD40L def. mice.
- Panels g andy represent CC; h and k, H; and i and /, EC.
- Scale bar denotes 100 ⁇ m (calculated for each panel), m, percentage of microgliosis and n, astrocytosis (mean + 1 SEM) were calculated by quantitative image analysis, and percentage reduction for each brain region is indicated.
- the t-Test for independent samples revealed significant between-groups differences for each brain region examined in m and « (p ⁇ .001 for each comparison).
- FIGS 2a-2g Congophilic amyloid deposits are markedly reduced in TgAPP s /CD40L def. mice by 16 months of age.
- Panels a-f axe representative 10X bright- field photomicrographs of mouse brain sections stained with congo red. The left column represents sections from TgAPP sw mice, and sections shown on the right were taken from TgAPP sw /CD40L def. mice.
- Panels ⁇ and d represent cingulate cortices (CC); b and e, hippocampi; and c and/, enthorinal cortices (EC). Scale bar denotes 100 ⁇ m (calculated for each panel).
- FIGS 3a-3h Morphometric analysis of A ⁇ plaques in TgAPP svv /CD40L def. mice versus TgAPP sw mice.
- Panels ⁇ -f are representative lOx bright-field photomicrographs of mouse brain sections (at 16 months of age) stained with anti-A ⁇ antibody.
- the left column represents sections from TgAPP sw mice, and sections shown on the right were taken from TgAPP s /CD40L def. mice.
- Panels ⁇ and d represent cingulate cortices (CC); b and e, hippocampi (H); and c and / enthorinal cortices (EC). Scale bar denotes 100 ⁇ m (calculated for each panel).
- Figures 4a-4g Reduced thioflavin S plaques in PSAPP mice treated with anti-CD40L antibody.
- Panels are 20x bright-field photomicrographs taken from 8-month-old PSAPP mice that received anti-CD40L antibody or isotype-matched control IgG antibody, a- f, mouse brain sections stained with thioflavin S; left column shows sections from isotype- matched IgG-treated mice, and sections shown in the right column were taken from anti- CD40L antibody-treated mice.
- Panels a and d were taken from cingulate cortices (CC); b and e, hippocampi (H); and c and f, entorhinal cortices (EC), g, percentages of thioflavin S- staining ⁇ -amyloid plaques (mean + 1 SEM) were quantified by image analysis, and percentage reduction for each brain region is indicated.
- the t-Test for independent samples revealed significant between-groups differences for each brain region examined in g (p ⁇ .001 for each comparison).
- FIGS 5a-5e CD40L modulates APP processing in vivo and in vitro.
- Brain homogenates were prepared from 12-month-old Tg APP SW , Tg APP SW /CD40L deficient (def.), control IgG-treated PSAPP, and anti-CD40L antibody-treated PSAPP animals. Representative lanes are shown from each mouse group, a, Western immunoblot by antibody 369 against the cytoplasmic tail of APP reveals holo APP, and two bands corresponding to C99 ( ⁇ -CTF) and C83 ( ⁇ -CTF) as indicated (top panel).
- the t-Test for independent samples revealed significant differences for each comparison (p ⁇ .001).
- Cell lysates and conditioned media were prepared from N2a cells over-expressing human APP and treated with 2 ⁇ g/mL of heat-inactivated CD40L (control) or CD40L protein (CD40 ligation) at the time points indicated, d, C-terminal fragments of APP were analyzed in cell lysates by Western immunoblot using antibody 369.
- FIGS 7A-7E Phospho-t ⁇ w in situ by antibody pS202.
- Figures 8A and 8C are from the neocortex and Figures 8B and 8D are from the hippocampus.
- (*) indicates A ⁇ plaques. Quantitative analysis of pooled date is shown in Figure 8E.
- the subject invention provides methods of treating neuronal inflammation, brain injury, tauopathies, or amyloidogenic diseases, comprising the administration of therapeutically effective amounts of a composition comprising a carrier and an agent that interferes with CD40L/CD40R signaling pathway to an individual afflicted with neuronal inflammation, brain injury, tauopathies, or an amyloidogenic disease.
- a composition comprising a carrier and an agent that interferes with CD40L/CD40R signaling pathway to an individual afflicted with neuronal inflammation, brain injury, tauopathies, or an amyloidogenic disease.
- the phrase "interferes with CD40L/CD40R signaling pathway” can be construed as disrupting the binding or association of CD40L with its cognate receptor, e.g., CD40R or interfering with the trimerization of CD40R.
- the phrase can be construed as disrupting the signaling pathway upstream or downstream of CD40L/CD40R binding.
- tauopathies are to be treated, agents reduce
- CD40 ligand refers to native, recombinant or synthetic forms of the molecule.
- Native, recombinant, or synthetic forms of CD40L can contain amino acid substitutions, additions, or deletions that do not affect the ability of the ligand to bind to the CD40 receptor (CD40R); in certain embodiments CD40LV bind to CD40R, are unable to activate the CD40R, and block the binding of native CD40L (e.g., CD40L having the naturally occurring amino acid sequence and the ability to activate CD40R).
- tauopathies include frontotemporal dementia, frontotemporal dementia with Parkinsonism, frontotemporal lobe dementia, pallidopontonigral degeneration, progressive supranuclear palsy, multiple system tauopathy, multiple system tauopathy with presenile dementia, Wilhelmsen-Lynch disease, disinhibition-dementia-parkinsonism-amytrophy complex, Pick's disease, or Pick's diseaselike dementia.
- Amyloidogenic diseases include, but not limited to, scrapie, transmissible spongioform encephalopathies (TSE's), hereditary cerebral hemorrhage with amyloidosis Icelandic-type (HCHWA-I), hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D), familial Mediterranean fever, familial amyloid nephropathy with urticaria and deafness (Muckle-Wells syndrome), myeloma or macroglobulinernia-associated idopathy associated with amyloid, familial amyloid polyneuropathy (Portuguese), familial amyloid cardiomyopathy (Danish), systemic senile amyloidosis, familial amyloid polyneuropathy (Iowa), familial amyloidosis (Finnish), Gerstmann-Staussler-Scheinker syndrome, medullary carcinoma of thyroid, isolated atrial amyloid, I
- terapéuticaally effective amounts is to be construed as an amount of a composition that confers an improvement in the condition of an individual treated according to the methods taught herein.
- improvements for an individual include, improvements in quality of life and/or memory, reductions in the size and/or number of amyloid plaques, reduction in ⁇ -amyloid burden, reduction of congophilic ⁇ -amyloid deposits, reduction of reactive gliosis, microgliosis, and/or astrocytosis, an improvement in the symptoms with which an individual presented to a medical practitioner (e.g., reductions in the severity of symptoms with which the individual presented), or reduction of other ⁇ -amyloid associated pathologies.
- An "agent that interferes with the interaction of CD40L and CD40R” includes, and is not limited to, soluble CD40R, antibodies that bind to CD40L and block its interaction with CD40R, antibodies that bind to CD40R and block ligand binding to the receptor, soluble CD40LV that bind to CD40R, but fail to activate the receptor, agents that reduce or inhibit the trimerization of CD40R, interfering RNA (dsRNA or RNAi) that suppresses or reduces the levels CD40R expression, antisense RNA to CD40R (in amounts sufficient to suppress or reduce the levels of CD40R expression), RNAi that reduces the levels or amounts of amyloid- ⁇ (A ⁇ ) protein that is expressed and that block or suppresses/reduces the ability of A ⁇ to induce CD40R expression, antibodies that bind to A ⁇ and block or suppress/reduce its ability to induce CD40R expression.
- Antibodies that bind to CD40R can agonize or, preferably, antagonize the function of
- Antisense technology can also be used to interfere with the CD40L/CD40R signaling pathway.
- the transformation of a cell or organism with the reverse complement of a gene encoded by a polynucleotide exemplified herein can result in strand co-suppression and silencing or inhibition of a target gene, e.g., A ⁇ , CD40L, or CD40R.
- Therapeutic protocols and methods of practicing antisense therapies for the modulation of CD40R are well-known to the skilled artisan (see, for example, U.S. Patent Nos. 6,197,584 and 6,194,150, each of which is hereby incorporated by reference in its entirety).
- RNAi or dsRNA-mediated interference RNAi or dsRNA-mediated interference
- dsRNA typically comprises a polynucleotide sequence identical or homologous to a target gene (or fragment thereof) linked directly, or indirectly, to a polynucleotide sequence complementary to the sequence of the target gene (or fragment thereof).
- the dsRNA may comprise a polynucleotide linker sequence of sufficient length to allow for the two polynucleotide sequences to fold over and hybridize to each other; however, a linker sequence is not necessary.
- the linker sequence is designed to separate the antisense and sense strands of RNAi significantly enough to limit the effects of steric hindrances and allow for the formation of dsRNA molecules and should not hybridize with sequences within the hybridizing portions of the dsRNA molecule.
- one method for treating amyloidogenic diseases comprises the use of materials and methods utilizing double-stranded interfering RNA (dsRNAi), or RNA-mediated interference (RNAi) comprising polynucleotide sequences identical or homologous to CD40L and/or CD40R.
- dsRNAi double-stranded interfering RNA
- RNAi RNA-mediated interference
- RNA containing a nucleotide sequence identical to a fragment of the target gene is preferred for inhibition; however, RNA sequences with insertions, deletions, and point mutations relative to the target sequence can also be used for inhibition.
- Sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group).
- the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a fragment of the target gene transcript.
- RNA may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro.
- a regulatory region e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation
- the promoters may be known inducible promoters such as baculovirus. Inhibition may be targeted by specific transcription in an organ, tissue, or cell type.
- RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
- RNA may be chemically or enzymatically synthesized by manual or automated reactions.
- the RNA may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6).
- a cellular RNA polymerase or a bacteriophage RNA polymerase e.g., T3, T7, SP6.
- T3, T7, SP6 bacteriophage RNA polymerase
- the RNA may be purified prior to introduction into the cell.
- RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof.
- the RNA may be used with no, or a minimum of, purification to avoid losses due to sample processing.
- the RNA may be dried for storage or dissolved in an aqueous solution.
- the solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
- dsRNAi can be targeted to an entire polynucleotide sequence, such as the CD40R, CD40L, or A ⁇ .
- Preferred RNAi molecules of the instant invention are highly homologous or identical to the polynucleotides encoding CD40R, CD40L, or A ⁇ .
- the homology may be greater than 70%, preferably greater than 80%, more preferably greater than 90% and is most preferably greater than 95%.
- Fragments of genes can also be utilized for targeted suppression of gene expression. These fragments are typically in the approximate size range of about 20 consecutive nucleotides of a target sequence. Thus, targeted fragments are preferably at least about 15 consecutive nucleotides. In certain embodiments, the gene fragment targeted by the RNAi molecule is about 20-25 consecutive nucleotides in length. In a more preferred embodiment, the gene fragments are at least about 25 consecutive nucleotides in length. In an even more preferred embodiment, the gene fragments are at least 50 consecutive nucleotides in length. Various embodiments also allow for the joining of one or more gene fragments of at least about 15 nucleotides via linkers. Thus, RNAi molecules useful in the practice of the instant invention can contain any number of gene fragments joined by linker sequences.
- Nucleotide sequences for CD40R, CD40L, and A ⁇ are known in the art and can be obtained from patent publications, public databases containing nucleic acid sequences, or commercial vendors.
- RNAi molecules can have a whole number value ranging from 15 consecutive nucleotides to one nucleotide less than the full length polynucleotide.
- the dsRNA molecules of the invention may be introduced into cells with single stranded (ss) RNA molecules which are sense or anti- sense RNA derived from the nucleotide sequences disclosed herein.
- ssRNA and dsRNA molecules are well-known to the skilled artisan and includes transcription of plasmids, vectors, or genetic constructs encoding the ssRNA or dsRNA molecules according to this aspect of the invention; electroporation, biolistics, or other well- known methods of introducing nucleic acids into cells may also be used to introduce the ssRNA and dsRNA molecules of this invention into cells.
- the subject invention provides methods for the treatment of internal organ diseases related to amyloid plaque formation, including plaques in the heart, liver, spleen, kidney, pancreas, brain, lungs and muscles comprising the administration of therapeutically effective amounts of a composition comprising a carrier and an agent that interferes with the CD40L/CD40R signaling pathway to an individual in need of such treatment.
- the present invention provides assays for identifying small molecules or other compounds capable of modulating CD40R/CD40L pathways.
- the assays can be performed in vitro using non-transformed cells, immortalized cell lines, recombinant cell lines, transgenic cells, transgenic cell lines, or transgenic animals and cells/cell lines derived therefrom.
- Transgenic animals suitable for use in the subject invention include transgenic worms, transgenic flies, transgenic mice.
- cells and cell lines can be of human or other animal origin.
- the assays can be used to examine the effects of small molecules or other compounds on with neuronal inflammation, brain injury, tauopathies, or an amyloidogenic disease.
- the small molecules or other compounds are tested for the ability to elicit an improvement in the condition of an individual to be treated according to the methods taught herein.
- cells are examined for decreased inflammation, other suitable changes in or markers that are followed by the skilled artisan.
- the subject invention provides in vivo methods of identifying small molecules or other compounds capable of modulating CD40R/CD40L signaling pathways comprising the administration of such compounds to individuals (e.g., human volunteers or murine models (such as those taught herein)) and examining the individuals for an improvement in the condition of an individual treated according to the methods taught herein.
- the subject invention also provides therapeutic compounds or small molecules and compositions comprising a carrier and said therapeutic compounds or small molecules.
- the carrier is a pharmaceutically acceptable carrier or diluent.
- compositions containing therapeutic compounds and/or small molecules can be administered to a patient in a variety of ways including, for example, parenterally, orally or intraperitoneally.
- Parenteral administration includes administration by the following routes: intravenous, intramuscular, interstitial, intra-arterial, subcutaneous, intraocular, intracranially, intraventricularly, intrasynovial, transepithelial, including transdermal, pulmonary via inhalation, opthalmic, sublingual and buccal, topical, including ophthalmic, dermal, ocular, rectal, and nasal inhalation via insufflation or nebulization.
- Compounds or small molecules that are orally administered can be enclosed in hard or soft shell gelatin capsules, or compressed into tablets. Active compounds or small molecules can also be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, sachets, lozenges, elixirs, suspensions, syrups, wafers, and the like.
- the pharmaceutical composition comprising the active compounds can be in the form of a powder or granule, a solution or suspension in an aqueous liquid or non- aqueous liquid, or in an oil-in-water or water-in-oil emulsion.
- the tablets, troches, pills, capsules and the like can also contain, for example, a binder, such as gum tragacanth, acacia, corn starch or gelating, excipients, such as dicalcium phosphate, a disintegrating agent, such as corn starch, potato starch, alginic acid and the like, a lubricant, such as magnesium stearate, and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent.
- a binder such as gum tragacanth, acacia, corn starch or gelating
- excipients such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, lactose or saccharin, or a flavoring agent.
- tablets, pills, or capsules can be coated with shellac, sugar or both.
- a syrup or elixir can contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic.
- the active compound can be incorporated into sustained-release preparations and formulations.
- the active compounds can be administered to the CNS, parenterally or intraperitoneally.
- Solutions of the compound as a free base or a pharmaceutically acceptable salt can be prepared in water mixed with a suitable surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative and/or antioxidants to prevent the growth of microorganisms or chemical degeneration.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form must be sterile and must be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size (in the case of a dispersion) and by the use of surfactants.
- a coating such as lecithin
- surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars or sodium chloride.
- Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and any of the other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and the freeze drying technique.
- compositions which are suitable for administration to the nose or buccal cavity include powder, self-propelling and spray formulations, such as aerosols, atomizers and nebulizers.
- the therapeutic compounds of this invention can be administered to a mammal alone or in combination with pharmaceutically acceptable carriers or as pharmaceutically acceptable salts, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
- compositions can also contain other therapeutically active compounds which are usually applied in the treatment of the diseases and disorders discussed herein. Treatments using the present compounds and other therapeutically active compounds can be simultaneous or in intervals.
- Tg APPsw mice manifest prominent astrocytosis and microgliosis and develop amyloid deposits comparable to human senile plaques by 16 months of age (Irizarry et al, "APPSw transgenic mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in CA1," J. Neuropathol. Exp. Neurol. (1997) 56:965-73).
- CD40L deficiency might oppose gliosis in Tg APP SW mice.
- CDl lb a marker of activated microglia
- GFAP glial fibrillary acidic protein
- TNF- ⁇ an activated microglial marker that we have shown is secreted after A ⁇ and CD40L challenge (Tan et al, "Microglial activation resulting from CD40- CD40L interaction after beta-amyloid stimulation," Science (1999) 286:2352-55) protein levels by Western immunoblot revealed a statistically significant (p ⁇ .001) 64% reduction in Tg APP S /CD40L def. mice compared to Tg APP S mice (mean TNF- ⁇ to actin ratio ⁇ 1 SEM: Tg APP sw mice, .247 ⁇ .02; control littermates, .13 + .01; Tg APP SW /CD40L def. mice, .09 + .01; CD40L def. mice, .09 ⁇ .02), providing further evidence of reduced inflammation in TgAPP sw /CD40L def. mouse brains.
- mice for the brain regions examined differences that were statistically significant (mean % + 1 SEM; 41% reduction in cingulate cortex: Tg APP SW , 1-74 ⁇ .22; Tg APP SW /CD40L def., 1.02 + .10, p ⁇ .05; 46% reduction in entorhinal cortex: Tg APP SW , 1.12 ⁇ .16; Tg APP SW /CD40L def., .60 ⁇ .06, p ⁇ .001; 51% reduction in hippocampus: Tg APP SW , -79 + .08; Tg APP SW /CD40L def, .39 + .08, p ⁇ .001).
- mice was not complete, we hypothesized that interrupting CD40R-CD40L signaling might specifically mitigate formation of the mature, congophillic subset of A ⁇ plaques. Strikingly, data show between 78 and 86% reduction in congophilic plaques in Tg APP SW /CD40L def. mice (Fig. 2). Morphometric analysis of anti-A ⁇ antibody immunoreactive A ⁇ plaques at this age corroborates these data, showing a similar magnitude of reduction in large (> 50 ⁇ m) and medium-sized (between 25 and 50 ⁇ m) A ⁇ plaque subsets in the neocortices and hippocampi of Tg APP SW /CD40L def. mice (Fig. 3).
- CD40L deficient mice are the C57BL/6 background and were constructed as previously described (Xu et al, “Mice deficient for the CD40 ligand,” Immunity (1994) 1:423-31).
- Tg APP SW mice are the 2576 line crossed with C57B6/SJL as previously described (Hsiao et al, "Age-related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins," Neuron (1995) 15:1203-18).
- CD40L deficient mice with Tg APP SW transgenic mice and characterized first and second filial offspring by polymerase chain reaction-based genotyping for the mutant APP construct (to examine Tg APP SW status) and neomycin selection vector (to type for CD40L deficiency), followed by Western blot for brain APP and splenic CD40L protein, respectively.
- mice The animals that we then studied at 12 and 16 months of age were Tg APP SW /CD40L deficient (Tg APP SW /CD40L def.; 12 months: 3 female, 16 months: 3 female/1 male), non-Tg APP SW /CD40L deficient (CD40L def.; 12 months: 3 female, 16 months: 3 female/1 male), Tg APP SW /CD40L wild-type (Tg APP SW ; 12 months: 3 female, 16 months: 2 female/ 1 male), and non-Tg APP SW /CD40L wild-type control littermate mice (Control; 12 months: 3 female, 16 months: 2 female/1 male).
- mice were anesthetized with isofluorane and transcardinally perfused with ice- cold physiological saline containing heparin. Brains were rapidly dissected and quartered using a mouse brain sheer (Muromachi Kikai Co., Tokyo, Japan). The first and second anterior quarters were homogenized for Western blot analyses, and the third and fourth posterior quarters were used for microtome or cryostat sectioning. For microgliosis analysis, brains were quick-frozen at -80°C, and for A ⁇ immunohistochemistry, congo red staining, and astrocytosis, brains were immersed in 4% paraformaldehyde at 4°C overnight, and routinely processed in paraffin.
- rabbit anti-cow GFAP antibody (1:500; DAKO, Carpinteria, CA)
- rabbit anti-human amyloid- ⁇ antibody (1:100; Sigma, Hercules, MO)
- rat anti-mouse CDl lb antibody (1:200; CALTAG LABORATOIRES, Burlingame, CA).
- Images were acquired from an Olympus BX60 microscope with an attached CCD video camera system (Olympus, Tokyo, Japan), and video signal was routed into a Windows 98SETM PC via an AG5 averaging flame grabber (Scion Corporation, Frederick, MD) for quantitative analysis using Image-Pro software (Media Cybernetics, MD).
- Mouse brains (Control, Tg APP SW , CD40L def., and Tg APP SW /CD40L def.) were isolated under sterile conditions on ice and placed in ice-cold lysis buffer (containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerolphosphate, 1 mM Na 3 VO 4 , 1 ⁇ g/mL leupeptin, and 1 mM PMSF).
- ice-cold lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerolphosphate, 1 mM Na
- TgAPP SW mice with animals deficient in CD40L (TgAPP sw /CD40L def.) (Tan et al, "Microglial activation resulting from CD40- CD40L interaction after beta-amyloid stimulation," Science (1999) 286:2352-55). [0062] In order to determine if genetic disruption of CD40L could produce diminished A ⁇ / ⁇ -amyloid pathology in Tg APP SW /CD40L def.
- mice we evaluated this pathology by four strategies: anti-A ⁇ antibody immunoreactivity (conventional " ⁇ -amyloid burden” analysis), A ⁇ sandwich enzyme-linked immunoabsorbance assay (ELISA), congo red staining, and ⁇ -amyloid plaque morphometric analysis. While 12-month old Tg APP SW mice had minimal ⁇ -amyloid plaque loads ( ⁇ 2 plaques per section examined), ⁇ -amyloid plaques were not detectable in age-matched Tg APP SW /CD40L def. mice.
- anti-A ⁇ antibody immunoreactivity conventional " ⁇ -amyloid burden” analysis
- ELISA sandwich enzyme-linked immunoabsorbance assay
- congo red staining congo red staining
- ⁇ -amyloid plaque morphometric analysis While 12-month old Tg APP SW mice had minimal ⁇ -amyloid plaque loads ( ⁇ 2 plaques per section examined), ⁇ -amyloid plaques were not detectable in age-matched Tg APP SW /CD40L def. mice.
- TgAPP SW /CD40L def Sixteen (16)- month-old TgAPP sw mice had typical ⁇ -amyloid load (Irizarry et al, "APPSw transgenic mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in CA1," Neuropathol. Exp. Neurol. (1997) 56:965-73), up to 51% diminution of ⁇ -amyloid burden was evident in Tg APP SW /CD40L def.
- mice as our data show a 78% (H) to 86% (CC) reduction compared to Tg APP SW mice.
- morphometric analysis of anti-A ⁇ antibody immunoreactive ⁇ -amyloid plaques at this age showed a reduction in large (> 50 ⁇ m) and medium-sized (between 25 and 50 ⁇ m) ⁇ -amyloid plaque subsets in their neocortices and hippocampi.
- Analysis of total APP by Western immunoblot did not reveal a significant difference between these mice (mean APP to actin ratio ⁇ 1 SEM; Tg APP SW mice, 1.16 ⁇ .06; Tg APP SW /CD40L def. mice, 1.15 ⁇ .04), suggesting that the observed reduction reduction of A ⁇ / ⁇ -amyloid in Tg APP SW /CD40L def. mice was not due to reduced APP production.
- Anti-CD40L antibody was administered to a transgenic mouse model of AD.
- PSAPP M146L PS1 mutations
- Anti-CD40L antibody was administered based on a treatment schedule previously described, which depletes CD40L in mice (Schonbeck et al, "Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice,” Proc. Natl. Acad. Sci. U S A (2000) 97:7458-63). At 8 months of age ⁇ -amyloid plaques appeared more diffuse in PSAPP mice that received anti-CD40L antibody treatment. Results revealed between 61% (H) and 74% (EC) reduction in ⁇ -amyloid plaques in PSAPP mice treated with anti-CD40L antibody versus isotype-matched control antibody.
- Tg APP SW /CD40L def. animals had a marked decrease of ⁇ -CTF relative to ⁇ - CTF.
- ⁇ -CTF was under-represented relative to ⁇ -CTF in animals that received non-relevant control IgG antibody (IgG-treated PSAPP mice did not differ from non-treated PSAPP animals, data not shown). This is consistent with the generation of excess A ⁇ / ⁇ -amyloid in these animals.
- PSAPP mice that received anti-CD40L antibody manifested a shift in APP CTFs such that the ratio of ⁇ -CTF to ⁇ -CTF was markedly decreased compared to controls.
- N2a cell line was established that stably overexpresses (by ⁇ 3-fold) the human wild-type APP-751 transgene (Xia et al, "Enhanced production and oligomerization of the 42-residue amyloid beta-protein by Chinese hamster ovary cells stably expressing mutant presenilins," J. Biol Chem. (1997) 272:7977-82).
- CD40L treatment of these cells results in a time-dependent decrease in ⁇ -CTF by Western blot. To confirm whether this reduction in ⁇ -CTF might be associated with amyloidogenic processing of APP, we measured secreted A ⁇ in conditioned media.
- Results show a time-dependent increase in both A ⁇ -4 o and A ⁇ i- ⁇ levels, which is inversely related to ⁇ -CTF levels.
- CD40L is able to directly promote amyloidogenic APP processing in neurons or neuron-like cells. Reducing the availability of CD40L in vivo has the opposite effect of adding CD40L in vitro on APP processing, both suggesting that CD40L regulates secretase cleavage of APP.
- the vast majority of cases of AD are associated with accumulation of A ⁇ from a normal APP sequence, the observation that the processing of normal APP can be pushed towards amyloidogenicity by CD40L is of interest.
- AD Alzheimer's disease
- CD40L-bearing astrocytes an excess of CD40L-bearing astrocytes occurs (Calingasan etal, "Identification of CD40 ligand in Alzheimer's disease and in animal models of Alzheimer's disease and brain injury," Neurobiol. Aging (2002) 23:31-9), and either membrane-bound or secreted forms of CD40L (Schonbeck et al, "The CD40/CD154 receptor/ligand dyad,” Cell Mol Life Sci. (2001) 58:4-43) could influence cerebral APP processing towards A ⁇ formation.
- mice are the C57BL/6 background constructed as previously described (Xu etal, “Mice deficient for the CD40 ligand,” Immunity (1994) 1:423-31).
- Tg APP SW mice are the 2576 line crossed with C57B6/SJL as previously described (Hsiao et al, "Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice,” Science (1996) 274:99-102).
- CD40L deficient mice were crossed with Tg APP SW transgenic mice and characterized offspring by polymerase chain reaction- based genotyping for the mutant APP construct (to examine Tg APP SW status) and neomycin selection vector (to type for CD40L deficiency), followed by Western blot for brain APP and splenic CD40L protein, respectively.
- mice The animals that we studied at 12 and 16 months of age were Tg APP SW /CD40L deficient (Tg APP SW /CD40L def; 12 months: 3 female, 16 months: 3 female/1 male), non-Tg APP SW /CD40L deficient (CD40L def; 12 months: 3 female, 16 months: 3 female/1 male), Tg APP SW /CD40L wild-type (Tg APP SW ; 12 months: 3 female, 16 months: 2 female/1 male), and non-Tg APP SW /CD40L wild-type control littermate mice (Control; 12 months: 3 female, 16 months: 2 female/1 male).
- PSAPP were bred by crossing Tg APP SW with PS1 Ml 467 mice as previously described (Holcomb etal, "Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes," Nat. Med. (1998) 4:97-100). A total of 10 PSAPP mice were used in this study, and 5 mice (3 female/2 male) received anti-CD40L IgG antibody (MR1), while the remaining 5 (2 female/3 male) received isotype-matched control IgG antibody. Beginning at 8 weeks of age, PSAPP mice were i.p.
- mice were anesthetized with isofluorane and transcardinally perfused with ice- cold physiological saline containing heparin. Brains were rapidly dissected and quartered using a mouse brain sheer (Muromachi Kikai Co., Tokyo). The first and second anterior quarters were homogenized for Western blot analyses, and the third and fourth posterior quarters were used for microtome or cryostat sectioning. For microgliosis analysis, brains were quick-frozen at -80°C, and for ⁇ -amyloid immunohistochemistry, congo red staining, and astrocytosis, brains were immersed in 4% paraformaldehyde at 4°C overnight, and routinely processed in paraffin.
- rabbit anti-cow GFAP antibody (1:500; DAKO), mouse anti-human amyloid- ⁇ antibody (4G8; 1:100; Signet), rabbit anti- human amyloid- ⁇ antibody (1:100; Sigma), and rat anti-mouse CDl lb antibody (1:200; Caltag Laboratories).
- ⁇ - amyloid, congo red, and thioflavin S burden data are reported as the percentage of immunolabeled area captured (positive pixels) divided by the full area captured (total pixels).
- ⁇ -amyloid plaque morphometric analysis diameters of ⁇ -amyloid plaques were calculated via quantitative image analysis and numbers of plaques falling into each diameter category were totaled. Each immunohistochemical analysis was performed by a single examiner (T.M. or T.T.). Image analysis was performed prior to the revelation of sample identities.
- Mouse brains (Control, Tg APP SW , CD40L def, and Tg APP SW /CD40L def.) were isolated under sterile conditions on ice and placed in ice-cold lysis buffer (containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerolphosphate, 1 mM Na 3 VO 4 , 1 ⁇ g/mL leupeptin, and 1 mM PMSF).
- ice-cold lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerolphosphate, 1 mM Na 3
- a ⁇ - o, and total A ⁇ (estimated by summing and A ⁇ j -42 enzyme-linked immunosorbent assay (ELISA) kits (QCB) in accordance with the manufacturer's instruction, except that standards were diluted such that the final concentration included 0.5 M guanidine buffer.
- Total protein was quantified in brain homogenates using the Bio-Rad protein assay (Bio-Rad); thus, ELISA values are reported as ng of A ⁇ i-x/wet g of brain.
- conditioned media from human APP-overexpressing N2a cells was collected and analyzed at a 1:1 dilution using the method described above, and values were reported as percentage of A ⁇ -X secreted relative to control.
- Membranes were then washed 3x for 5 min each in dH 2 0 and incubated for lh at ambient temperature with the appropriate HRP-conjugated secondary antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). All antibodies were diluted in TBS containing 5% (w/v) of non-fat dry milk. Blots were developed using the luminol reagent (Santa Cruz). Densitometric analysis was performed using the Fluor-S MultilmagerTM with Quantity OneTM software (Bio-Rad). Antibodies used for Western blot included antibody 369 (1:500, kindly provided by Dr. Sam Gandy), 6687 (1:1,000, kindly provided' by Dr. Harald Steiner), Chemicon anti-C-terminal APP antibody (1:500), BAM-10 (1:1000, Sigma), or actin (as an internal reference control, 1:1000, Roche, Germany).
- Immunohistochemical staining was performed in accordance with the manufacturer's instruction using the VECTASTATN® Elite avadin biotin complex (ABC) kit (Vector Laboratories, Burlingame, CA).
- the primary antibodies that were employed were anti- phospho-t ⁇ w SI 99 (1:50) and anti-phospho-taM S202 (1:200) (both antibodies were obtained from BioSource International, Camarillo. CA).
- Slides were permanently mounted and viewed under bright-field using an Olympus BX-60 microscope. [0075] Image analysis.
- Bright-field images were acquired from an Olympus BX-60 microscope with an attached MagnaFireTM camera, and video signal was routed into a Windows 98SETM PC for quantitative analysis using Image-Pro software (Media Cybernetics, Silver Spring, MD). Images of five 5 ⁇ m sections (150 ⁇ m apart) through each anatomic region of interest (hippocampus or cortical areas) were captured and a threshold optical density was obtained that discriminated staining from background. Manual editing of each field was used to eliminate artifacts. Positive immunolabeled area was determined by dividing the percentage of immunolabeled area captured (positive pixels) by the full area captured (total pixels). Image analysis was performed in a blind fashion prior to the revelation of sample identities.
- the t-Test for independent samples revealed significant differences between Tg APP SW and Tg APP SW /CD40L def. mice for the neocortex (p ⁇ .01) and the hippocampus (p ⁇ .05). Immunostaining was also performed using antibody pS202. The pattern of immunoreactivity for this antibody was quite different from that of pS199, as pS202 revealed a punctate staining pattern within the area delineated by the ⁇ -amyloid deposit, while pS202 -positive neurons surrounding the ⁇ -amyloid deposit were few in number in both the neocortex and the hippocampus of Tg APP SW mice.
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STUBER ECKHARD ET AL: "Blocking the CD40L-CD40 interaction in vivo specifically prevents the priming of T helper 1 cells through the inhibition of interleukin 12 secretion" BIOSIS, 1996, XP002189392 * |
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