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WO2018172540A1 - Procédé de prédiction de la progression de la maladie d'alzheimer - Google Patents

Procédé de prédiction de la progression de la maladie d'alzheimer Download PDF

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Publication number
WO2018172540A1
WO2018172540A1 PCT/EP2018/057511 EP2018057511W WO2018172540A1 WO 2018172540 A1 WO2018172540 A1 WO 2018172540A1 EP 2018057511 W EP2018057511 W EP 2018057511W WO 2018172540 A1 WO2018172540 A1 WO 2018172540A1
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WIPO (PCT)
Prior art keywords
disease
patient
alzheimer
neutrophils
predetermined reference
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PCT/EP2018/057511
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English (en)
Inventor
Carole ELBIM
Yuan Dong
Guillaume Dorothee
Marie SARAZIN
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Sorbonne Université
Université Paris-Sud
Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives (Cea)
Centre National De La Recherche Scientifique (Cnrs)
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Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Sorbonne Université, Université Paris-Sud, Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives (Cea), Centre National De La Recherche Scientifique (Cnrs) filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Publication of WO2018172540A1 publication Critical patent/WO2018172540A1/fr

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to a method for predicting the rapidity of progression of
  • Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the neutrophils are not hyperactivated as compared to the predetermined reference.
  • AD Alzheimer's disease
  • intracellular neurofibrillary tangles chiefly comprised of abnormally folded tau protein and gliosis consisting of reactive microglia and astrocytes surrounding ⁇ -amyloid plaques.
  • gliosis consisting of reactive microglia and astrocytes surrounding ⁇ -amyloid plaques.
  • PMN Polymorphonuclear neutrophils
  • ROS reactive oxygen species
  • NETs neutrophil extracellular traps
  • PMNs are also important - - mediators of inflammation-induced injury and their improper activation may lead to an oxidative stress and exaggerated inflammatory reactions [Nemeth, 2012; Nathan, 2006].
  • the aim of the present study was to phenotypically and functionally characterize human circulating PMNs at different disease stages in AD. This study was performed in whole-blood conditions in order to minimize activation due to isolation procedures. This presents the advantage of taking into account the proinflammatory cytokine environment. The inventors also investigated associations between these indicators and rate of disease progression.
  • the present invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the neutrophils are not hyperactivated as compared to the predetermined reference.
  • the invention is defined by its claims.
  • a first aspect of the invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the neutrophils are not hyperactivated as compared to the predetermined reference.
  • the term "rapidity of progression of Alzheimer's disease (AD)” allows to discriminate AD patients into slow decliners (SD) or fast decliners (FD) based on the progression of the Clinical Dementia Rating (CDR) score after two years and/or the progression of the Mini-Mental State Examination (MMSE) score after one or two years.
  • SD Patients with Alzheimer's disease remain stable, showing unchanged CDR score, while patients with Alzheimer's disease declin (FD), with an increase of 0.5 or more of the CDR score (see for example Solomon PR et al, 1998).
  • CDR Clinical Dementia Rating
  • MMSE Mini-Mental State Examination
  • the neutrophils are the polymorphonuclear neutrophils (PMN).
  • the invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of polymorphonuclear neutrophils ii) comparing the activation state of the polymorphonuclear neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the polymorphonuclear neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the polymorphonuclear neutrophils are not hyperactivated as compared to the predetermined reference.
  • hypoactivated neutrophils or “hyperactivated polymorphonuclear neutrophils” denote a state of activation of these cells characterised by different parameters like an increased oxidative stress comprising an increased constitutive ROS (free radicals) production by resting PMNs and an increased capacity of PMNs to produce ROS in response to various stimuli), a decrease in L-selectin (CD62L) or FCyRIIIB (CD 16b) expression on resting PMN, an increased degranulation characterized by an higher CD1 lb and CD1 lc expression on resting PMNs or an increased percentage of apoptotic and necrotic PMNs as well as a decreased PMN survival in response to stimuli such as TNFa or TLR agonists.
  • an increased oxidative stress comprising an increased constitutive ROS (free radicals) production by resting PMNs and an increased capacity of PMNs to produce ROS in response to various stimuli
  • L-selectin CD62L
  • FCyRIIIB CD 16
  • the measurement of the oxidative stress is realized as described in the Materials & Methods parts and for example in the article Bass DA et al 2016.
  • the result of the oxydative stress will be expressed as mean fluorescence intensity (MFI) of Ethidium which is directly correlated to the amount of reactive oxygen species produced by PMNs.
  • MFI mean fluorescence intensity
  • the measurement of the decrease in L-selectin (CD62L) or FCyRIIIB (CD 16b) expression on resting PMN is realized as described in the Materials & Methods parts and for example in the article Campillo-Gimenez L et al, 2014.
  • the result will be expressed as mean fluorescence intensity (MFI) which correlated with the number of molecules of CD62L or CD 16b per PMN.
  • MFI mean fluorescence intensity
  • the measurement of the increased degranulation is realized as described in the Materials & Methods parts and for example in the article Campillo-Gimenez L et al, 2014.
  • the result will be expressed as mean fluorescence intensity (MFI) which correlated with the number of molecules of CD1 lb or CD1 lc per PMN.
  • MFI mean fluorescence intensity
  • the measurement of the increased percentage of apoptotic and necrotic PMNs is realized as described in the Materials & Methods parts and for example in the article Francois S. et al 2005. The results will be expressed as the percentage of apoptotic and necrotic PMNs among the total population of PMNs.
  • the measurement of the decreased PMN survival is realized as described in the Materials & Methods parts and for example in the article Francois S. et al 2005.
  • the results will be expressed as the percentage inhibition of PMN apoptosis.
  • the measured values will be interpreted by comparison with the reference interval (Rl ) defined as the central 95% of a reference population.
  • the reference population includes age-matched well-defined healthy individuals (reference individuals) (see for example J. Graham and A. Barker. Reference intervals. Clin Biochem Rev 2008 Aug; 29(Suppl 1): S93-S97).
  • Exclusion criteria in the reference population included acute or chronic inflammatory conditions, and corticoid or non-steroidal antiinflammatory drugs, known to modulate PMN phenotype and functions (see for example Campillo-Gimenez L et al, 2014).
  • Table 1 reference intervals and mean for each parameters used to evaluate the activation status of neutrophils.
  • At least one parameter indicated above can be used alone or combined with the others to determine the activation state of neutrophils.
  • sample denotes, blood, peripheral-blood, serum, plasma, urine or Cerebrospinal fluid (CSF).
  • CSF Cerebrospinal fluid
  • the term "patient” refers to an individual with symptoms of Alzheimer's disease. Patients with Alzheimer's disease are characterized by the following criteria: (i) progressive episodic memory impairment, characterized by a low free recall not normalized with semantic cueing [Sarazin et al., 2007 and Dubois et al., 2010]; (ii) absence of extrapyramidal signs; and (iii) CSF Alzheimer's disease profile, when available, defined as score 50.8, calculated with the formula amyloid- 42/[240 + (1.18 x T-tau)] [De Souza et al.,
  • the invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive PMN subsets ii) comparing the ratio between the senescent and the immunosuppressive PMN subsets at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the ratio between the senescent and the immunosuppressive PMN subsets is higher than its predetermined reference or predicting a slow progression when the ratio between the senescent and the immunosuppressive PMN subsets did not differ from its predetermined reference.
  • the term "senescent PMN subset” denote aged or senescent CXCR4 hlgh PMNs and are characterized by lower expression of CD62L, increased expression of ⁇ 2 integrins, as well as increased production of ROS and susceptibility of NET formation compared to the total circulating pool (see for example Zhang D. et al, 2015).
  • the term “immunosuppressive PMN subset” denote CD16 bright /CD62L dim PMNs, showing decreased adhesion properties and ROS production (see for example Pillay J. et al, 2012).
  • the measurement of the ratio between the senescent and the immunosuppressive PMN subsets is realized as described in the Materials & Methods parts and for example in the article Sauce D. et al, 2017.
  • the measured values will be interpreted by comparison with the reference interval (RI) defined as the central 95% of a reference population.
  • the reference population includes age- matched well-defined healthy individuals (reference individuals). Exclusion criteria in the - - reference population included acute or chronic inflammatory conditions, and corticoid or nonsteroidal anti-inflammatory drugs, known to modulate PMN phenotype and functions (see for example Campillo-Gimenez L et al, 2014) (see table 2).
  • Table 2 reference intervals and mean for the ratio between the senescent and the immunosuppressive PMN.
  • the inventors also show that the activation state of the neutrophils (or polymorphonuclear neutrophils) could be used to determine the severity of Alzheimer's disease that is to say if the patient has an Alzheimer's disease with dementia or not.
  • the invention relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the expression level determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower than its predetermined reference value.
  • the invention in another embodiment, relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive PMN subsets ii) comparing the ratio between the senescent and the immunosuppressive PMN subsets at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the ratio determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the ratio determined at step i) is lower than its predetermined reference value.
  • the invention in another embodiment, relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the level of circulating intravascular NETs ii) comparing the level of circulating intravascular NETs at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the level determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the level determined at step i) is lower than its predetermined reference value.
  • the parameters described above can be used to stratify patients suffering from Alzheimer's disease.
  • the patients can be stratified as: Alzheimer's disease with mild cognitive impairment also called prodromal Alzheimer's disease or Alzheimer's disease with dementia.
  • the inventors also showed that some proteins and particularly some cytokines could be used for predicting the severity of Alzheimer's disease.
  • the invention in another embodiment, relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the expression level of MMP-9 and the expression level of TIMP-1 ii) comparing the ratio determined at step i) with its predetermined reference value and iii) determining that the patient has a severe Alzheimer's disease when the ratio determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower than its predetermined reference value.
  • the invention relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the expression level of at least one protein selected in the group consisting in IL- ⁇ , IL-6, TNF- , IL-18, IL-8, IL17, IL-22 and IL10 ii) comparing the expression level determined at step i) with their predetermined reference values and iii) determining that the patient has a severe Alzheimer's disease when the expression level determined at step i) is higher than their predetermined reference values for at least one of the proteins IL- ⁇ , IL-6, TNF-a, IL-8 and IL17 or when the expression level determined at step i) is lower than their predetermined reference values for at least one of the proteins IL-18, IL-22 and IL10 or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower than their predetermined reference values for at least one of the proteins IL-
  • TNF- ⁇ , IL-8 and IL17 or when the expression level determined at step i) is higher than their predetermined reference values for at least one of the proteins IL-18 IL-22 and IL10.
  • severe Alzheimer's disease denotes that the patient suffer from an Alzheimer's disease with dementia.
  • a "not severe Alzheimer's disease is characterised by an Alzheimer's disease with mild cognitive impairment also called prodromal Alzheimer's disease.
  • MMP-9 for "Matrix metallopeptidase 9" also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB), has its general meaning in the art and denotes a matrixin, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the extracellular matrix.
  • the MMP9 gene encodes for a signal peptide, a propeptide, a catalytic domain with inserted three repeats of fibronectin type II domain followed by a C-terminal hemopexin-like domain.
  • An exemplary sequence for human MMP-9 protein is deposited in the UniProt database under accession numbers P14780.
  • TIMP-1 tissue inhibitors to the metalloproteinases-1 » denote a factor that forms complexes 1 : 1 with active MMP-9 and the MMP-9/TIMP-1 balance reflects the net proteolytic activity present in several physiological processes.
  • An exemplary sequence for human TIMP-1 protein is deposited in the UniProt database under accession numbers P01033.
  • II- 1 ⁇ for "Interleukin 1 beta” also known as leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, lymphocyte activating factor has its general meaning in the art and denotes a cytokine protein that in humans is encoded by the IL1B gene.
  • IL-1 interleukin- 1
  • IL-1 alpha IL-1 alpha
  • IL-1 beta this gene
  • IL- ⁇ precursor is cleaved by cytosolic caspase 1 (interleukin 1 beta convertase) to form mature IL- 1 ⁇ .
  • An exemplary sequence for human IL- 1 ⁇ protein is deposited in the UniProt database under accession numbers P01584.
  • IL-6 for "Interleukin 6" has its general meaning in the art denotes an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. Interleukin 6 is secreted by T cells and macrophages to stimulate immune response, e.g. during infection and after trauma, especially burns or other tissue damage leading to inflammation. IL-6 also plays a role in fighting infection, as IL-6 has been shown in mice to be required for resistance against bacterium Streptococcus pneumoniae. An exemplary sequence for human IL- 1 ⁇ protein is deposited in the UniProt database under accession numbers P05231. - -
  • TNF-a for "Tumor necrosis factor” has its general meaning in the art and denotes a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons.
  • cytokine cell signaling protein
  • NK cells NK cells
  • neutrophils neutrophils
  • mast cells eosinophils
  • neurons eosinophils
  • IL-18 for "Interleukin-18", also known as interferon-gamma inducing factor, has its general meaning in the art and denotes a protein which in humans is encoded by the IL18 gene.
  • the protein encoded by this gene is a proinflammatory cytokine.
  • An exemplary sequence for human IL-18 protein is deposited in the UniProt database under accession numbers Q 14116.
  • IL-8 for "Interleukin 8" (has its general meaning in the art and denotes a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells and endothelial cells. Endothelial cells store IL-8 in their storage vesicles, the Weibel-Palade bodies.
  • the interleukin-8 protein is encoded by the IL8 gene. IL-8 is initially produced as a precursor peptide of 99 amino acids long which then undergoes cleavage to create several active IL-8 isoforms. In culture, a 72 amino acid peptide is the major form secreted by macrophages.
  • An exemplary sequence for human IL-8 protein is deposited in the UniProt database under accession numbers P10145.
  • IL-17 for "Interleukin 17” has its general meaning in the art and denotes the founding member of a group of cytokines called the IL-17 family.
  • IL-17 shows high homology to viral IL-17 encoded by an open reading frame of the T-lymphotropic rhadinovirus Herpesvirus saimiri.
  • the IL-17 used is the IL-17 A.
  • An exemplary sequence for human IL-17A protein is deposited in the UniProt database under accession numbers Q 16552.
  • IL-22 for "Interleukin-22” has its general meaning in the art and denotes a member of a group of cytokines called the IL-10 family or IL-10 superfamily (including IL-19, IL-20, IL-24, and IL-26), a class of potent mediators of cellular inflammatory responses.
  • IL-10 family or IL-10 superfamily including IL-19, IL-20, IL-24, and IL-26
  • An exemplary sequence for human 11-22 protein is deposited in the UniProt database under accession numbers Q9GZX6.
  • IL-10 for "Interleukin 10" has its general meaning in the art and denotes an anti-inflammatory cytokine.
  • interleukin 10 is encoded by the IL10 gene.
  • IL-10 signals through a receptor complex consisting of two IL-10 receptor- 1 and two IL- 10 receptor 2 proteins. Consequently, the functional receptor consists of four IL-10 receptor - - molecules.
  • IL-10 binding induces STAT3 signalling via the phosphorylation of the cytoplasmic tails of IL-10 receptor 1 + IL-10 receptor 2 by JAKl and Tyk2 respectively.
  • An exemplary sequence for human IL- 10 protein is deposited in the UniProt database under accession numbers P22301.
  • the biomarkers described above can be used to stratify patients suffering from Alzheimer's disease.
  • the patients can be stratified as: Alzheimer's disease with mild cognitive impairment also called prodromal Alzheimer's disease or Alzheimer's disease with dementia.
  • the ratio MMP-9/TIMP-1 and the cytokines IL17 and IL18 can be used to stratify patients suffering from Alzheimer's disease.
  • Measuring the expression level of the proteins of the invention can be done by measuring the gene expression level of these proteins or by measuring the level of the proteins and can be performed by a variety of techniques well known in the art.
  • the expression level of a gene may be determined by determining the quantity of mR A in peripheral leucocytes populations.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the samples e.g., cell e ⁇ tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable - - label).
  • a "detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • catalysts such as enzymes
  • haptens that can be detected by antibody binding interactions
  • paramagnetic and magnetic molecules or materials paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • DBITC 4'-isothiocyanate
  • eosin and derivatives such as eosin and eosin isothiocyanate
  • erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate
  • ethidium fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfiuorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyros
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • a secondary emission of energy occurs of a frequency that corresponds to the handgap - - of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20132016, 1998; Chan et al, Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos.
  • quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • radioisotopes such as 3 H
  • metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
  • liposomes include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can be used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • enzymes for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can be used in a metallographic detection scheme.
  • SISH silver in situ hyhridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along - - with a water-soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • ISH In situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin,
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in - - this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in - - this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling - - may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • Expression level of a gene may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the stage of the patient, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • the level of the proteins of the invention may also be measured and can be performed by a variety of techniques well known in the art.
  • protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • ELISA ELISA
  • Detection of protein concentration in the sample may also be performed by measuring the level of the proteins of the invention.
  • the "level of the proteins” or the “proteins level expression” means the quantity or concentration of said proteins.
  • the "level of the proteins” means the level of fragments of the proteins.
  • the "level of the proteins” means the quantitative measurement of the proteins expression relative to a negative control.
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS). etc.
  • the reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
  • Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value.
  • concentration of the proteins of the invention refers to an amount or a concentration of a transcription product, for instance the proteins.
  • a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example.
  • relative units can be employed to describe a concentration.
  • concentration of proteins may refer to fragments of the proteins.
  • fragment of the proteins may also be measured.
  • the detection of the level of the proteins of the invention can be performed by flow cytometry.
  • the detection of the level of the proteins of the invention can be performed by luminex assays.
  • the detection of the level of the proteins of the invention can be performed by MesoScale Discovery assays.
  • the detection of the level of the proteins of the invention can be performed by nano-ELISA assays, or other ultrasensitive single molecule immunoassays, including Simoa technology.
  • Predetermined reference values used for comparison for the proteins of the invention may comprise "cut-off or "threshold" values that may be determined as described herein.
  • Each - - reference (“cut-off) value for the proteins expression of the invention may be predetermined by carrying out a method comprising the steps of
  • step e providing, for each sample provided at step a), information relating to the actual clinical status for the corresponding patient (severe AD or not);
  • the reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest.
  • the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.
  • the reference value (cut-off value) may be used in the present method to discriminate AD samples and therefore the corresponding patients.
  • Such predetermined reference values of expression level may be determined for any gene defined above.
  • kits for performing the methods of the invention comprise means for measuring the expression level of the proteins of the invention in the sample obtained from the patient.
  • kits may include probes, primers macroarrays or microarrays as above described.
  • the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled.
  • probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise - - hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • the kit of the invention may comprise amplification primers that may be pre- labelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • a second aspect of the invention relates to a method for monitoring the efficacy of a treatment of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the treatment is effective when the neutrophils are not hyperactivated as compared to the its predetermined reference or predicting that the treatment is not effective when the neutrophils are hyperactivated as compared to the its predetermined reference.
  • the invention in another embodiment, relates to a method for monitoring the efficacy of a treatment of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive PMN subsets ii) comparing the ratio between the senescent and the immunosuppressive PMN subsets at step i) with its predetermined reference and iii) predicting that the treatment is not effective when the ratio between the senescent and the immunosuppressive PMN subsets is higher than its predetermined reference or predicting that the treatment is effective when the ratio between the senescent and the immunosuppressive PMN subsets did not differ from its predetermined reference.
  • Another aspect of the invention relates to a method for stratifying patients as fast or slow progressor or as having a severe or not a severe Alzheimer's disease for designing a clinical trials to test new compound for the treatment of Alzheimer's disease by using the activation status of the neutrophils as determined in the invention and/or the biomarkers of the invention (the proteins of the invention).
  • a third aspect of the invention relates to a compound that reduces the presence or the activity of neutrophils in blood and/or in brain for use in the treatment of Alzheimer's disease in a patient with a rapid progression and/or a severe disease as described above.
  • a "compound that reduces the presence or activity of neutrophils in blood and/or in brain” modulates or interferes with at least one pathway of neutrophil trafficking - - or activation and is well described in the patent application WO 2015051152.
  • said compound may be an inhibitor, blocking or depleting agent, e.g. an antibody that depletes a targeted cell population, an agonist, or an antagonist of one or more of these pathways.
  • said compound may block the activity of a protein that acts in one or more of these pathways, e.g. a chemoattractant, a protein tyrosine kinase, an adhesion molecule, etc.
  • Pathways of interest for intervention by the methods of the invention include (i) depletion of neutrophil/myeloid cell populations systemically or locally in the brain; (ii) blocking neutrophils/myeloid cell adhesion and crawling; (iii) blocking transmigration and infiltration of neutrophils/myeloid cells into the brain; (iv) blocking cell-cell interactions between neutrophil/myeloid cells and endothelial cells and/or neural cells; (v) blocking neutrophil/myeloid cell extracellular-matrix interactions; (vi) reducing motility of neutrophils/myeloid cells in the brain parenchyma; (vii) blocking ⁇ -induced activation and adhesion of neutrophils/myeloid cells; (viii) blocking intracellular signalling controlling adhesion and activation; (ix) blocking neutrophil activation and/or degranulation; (x) blocking release of reactive oxygen species, proteases, cytokines, lipid mediators or other damaging agents from myeloid cells and/or neutrophils; (xi)
  • compound that targets adhesion molecules involved in neutrophils trafficking or extravasation including but not limited to: integrins and their ligands, e.g. ICAM- 1, LFA-1, CDl la, CDl lb, CDl lc, CD18, alpha-4 integrins and their ligands VCAM-1 and MAdCAM-1; CD49; E-, P- and L- selectin and their ligands, e.g. including but not limited to PSGL-1 , CD44, CD43, hyaluronan, glycolipids,
  • the compound inhibits the interaction between an adhesion molecule involved in neutrophils trafficking to the brain, and its ligand.
  • compound targeting protein tyrosine kinases included but not limited to, Syk, Abl, JAK3, Jak2, and BTK and MAPK; and/or PI3K.
  • further therapeutic active agent can be administered to the patient.
  • This active agent can be a cholinesterase inhibitor, the memantine, a N-methyl D- aspartate antagonist, a BACE1 inhibitor, a ⁇ secretase inhibitor, passive anti- ⁇ - - immunotherapy in the form of monoclonal anti- ⁇ antibodies, active anti- ⁇ immunotherapy, non-steroidal anti-inflammatory drugs, drugs enhancing ⁇ clearance, drugs inhibiting ⁇ aggregation, drugs reducing Tau phosphorylation, drugs inhibiting Tau aggregation, passive anti-Tau immunotherapy in the form of monoclonal anti-Tau antibodies, active anti-Tau immunotherapy, and APOE-related treatment approaches.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from the disease or medical condition.
  • the treatment may be administered to a subject having the medical disorder or who ultimately may acquire the disorder.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of the disease.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • a fourth aspect of the invention relates to a therapeutic composition
  • a therapeutic composition comprising compound that reduces the presence or activity of neutrophils in blood and/or in brain according to the invention for use in the treatment of Alzheimer's disease in a patient with a rapid progression and/or a severe disease as described above.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. - -
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intrathecal or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • FIGURES are a diagrammatic representation of FIGURES.
  • AD-MCI prodromal Alzheimer's disease group
  • AD-D Alzheimer's disease dementia group
  • CDR Clinical Dementia Rating
  • MMSE Mini-Mental State Examination
  • PiB Pittsburgh compound B
  • n number. °p ⁇ 0.05 compared to healthy controls (HC). *p ⁇ 0.05 compared to AD MCI.
  • C ROS production by stimulated PMNs was measured after pretreatment of whole-blood samples for 45 minutes with PBS, Pani3CSK 4 (TLR1/2 agonist, 1 ⁇ g/mL), or LPS (TLR4 agonist, 10 ng/ml), or TNF-a (TNF, 5 ng/ml) and incubation for 5 minutes with fMLP (10 "6 M); results are expressed as MFI.
  • D Quantification of circulating NETs was performed by MPO-DNA complex ELISA; the mean optical density was measured by capture ELISA and results are expressed as ng/ ⁇ DNA.
  • Results are expressed as the percentage inhibition of PMN apoptosis [1 - (% of total annexin V + /7-AAD " PMN in TLR agonist-treated sample/% of total annexin V + /7- AAD PMN in PBS-treated sample)] X 100.
  • CD62L and E CD l ib expression on PMN surface were measured after incubation of whole-blood samples for 45 minutes with PBS, or TLR1/2 agonist, or TLR4 agonist, or TNF-a; results are expressed as MFI.
  • PMNs from the AD prodromal group and the AD dementia group exhibit differential pro-inflammatory properties in relation with alterations of circulating neutrophils homeostasis.
  • E Whole-blood samples were incubated for 45 minutes at 4°C with FITC-anti-human CD 16, PE-anti-human CD 11c, Pe-Cy7-anti-human CD l ib, and APC-anti-human CD62L (BD). Results are expressed as the percentages of the CD16 bright /CD62L dim immunosuppressive PMN subset.
  • F Ratio between the senescent and the immunosuppressive subsets.
  • G Correlation between the percentage of the CXCR4 bright /CD62L dim PMN subset and ROS production by TLR4-agonist-primed PMNs.
  • Figure 4 Characterization of the activation state, chemotaxis, phagocytosis and apoptosis of PMNs from the AD prodromal group and the AD dementia group.
  • PMN hyperactivation state and imbalance of circulating senescent and immunosuppressive PMN subsets are associated with the rapidity of AD evolution.
  • Circulating levels of pro- and anti-inflammatory cytokines were measured from serum using Luminex assays. Correlation between MMSE and IL-8 circulating levels (B), and IL-17 circulating levels (C).
  • Circulating levels of MMP-9 (A) and TIMP-1 (B) were measured from serum using Luminex assays.
  • Alzheimer's disease Patients with Alzheimer's disease were included according to the following criteria: (i) progressive episodic memory impairment, characterized by a low free recall not normalized with semantic cueing ⁇ Sarazin, 2007 #204 ⁇ ⁇ Dubois, 2010 #205 ⁇ ; (ii) absence of extrapyramidal signs; and (iii) CSF Alzheimer's disease profile, when available, defined as score 50.8, calculated with the formula amyloid- 42/[240 + (1.18 x T-tau)] ⁇ de Souza, 2011 #206 ⁇ .
  • MMSE Mini-Mental State Examination
  • CDR Clinical Dementia Rating
  • TNF Tumor Necrosis Factor
  • TNF Tumor Necrosis Factor
  • TLR Toll Like Receptor
  • Pani3CSK4 TLR1/2 agonist, 1 ⁇ g/mL, Invivogen, San Diego, CA
  • LPS Lipopolysaccharide from E. coli serotype R515 (TLR4 agonist, lOng/mL, Alexis Biochemicals, San Diego, CA) for 45 minutes at 37°C.
  • whole-blood samples were incubated were incubated in 24-well tissue culture plates, for 3 or 5 hours, at 37°C with PBS, ⁇ 42 amyloid peptide (20 ⁇ ), IL-6 (1-100 ng/ml, R&D Systems), IL-8 (1-100 ng/ml, R&D Systems), TNFa (1-100 ng/ml, R&D Systems), IL-17 (1-lOOng/ml, R&D Systems) or IL-18 (1-lOOng/ml, R&D Systems) before staining.
  • the blood was then lysed with BD FACS lysing and cells were then resuspended with Cell Fix IX (BD Biosciences).
  • the percentage of PMN phagocytosing opsonized Escherichia Coli (E. coli) as well as the mean fluorescence intensity (MFI) was measured in whole blood by using the Phagotest Kit (Glycotope Biotechnology).
  • Whole blood samples ( ⁇ ) were incubated with opsonized FITC-conjugated E. coli bacteria (2xl0 7 per 20 ⁇ 1) for 10 minutes in a water bath at 37°C, the - - phagocytosis was then stopped in ice and erythrocytes are lysed before analysis by flow cytometry ⁇ Sauce, 2017 #152 ⁇ .
  • Superoxide anion (O2 " ) production by PMNs was measured using a flow cytometry based assay derived from the hydroethidine (HE) oxidation technique, as previously described ⁇ Campillo-Gimenez, 2014 #22 ⁇ .
  • Heparin-whole blood samples 500 ⁇ were loaded for 15 minutes with 1500ng/mL HE (Fluka, Buchs, Switzerland) at 37°C, and then incubated for 45 minutes with PBS or various stimuli, as described above. Samples were then treated with PBS or 10 "6 M fMLP (Sigma Chemical Co., St Louis, MO) for 5 minutes. Erythrocytes were lysed before analysis by flow cytometry.
  • PMN cell death in whole blood was quantified with annexin V and the impermeant nuclear dye 7-amino-actinomycin D (7-AAD).
  • Whole-blood samples 500 ⁇ were incubated in 24-well tissue culture plates, for 20 hours, at 37°C with PBS or various stimuli as described above. Samples were incubated with APC-anti-CD 15 antibodies (clone HI98, BD Biosciences), FITC-annexin V, and 7-AAD (BD Biosciences) as previously described ⁇ Campillo-Gimenez, 2014 #22 ⁇ and analyzed by flow cytometry.
  • MFI Mean Fluorescence Intensity
  • PMN phagocytic index % phagocytic PMN x MFI.
  • PMNs were identified as CD15 hlgh cells and 2> ⁇ 10 5 events were counted per sample. Results were expressed as the percentage of apoptotic (Annexin V + /7-AAD " ) and necrotic PMNs (AnnexinV77-AAD + ).
  • Chemo taxis was measured in Transwell plates (Corning Costar) containing 3- ⁇ pore- size polyvinylpyrrolidone-free polycarbonate filters ⁇ Berthelot, 2012 #153 ⁇ .
  • the lower well of - - each chamber received ⁇ of fMLP (10 "7 M), or IL-8 (25ng/ml) diluted in PBS plus 1% human serum albumin.
  • Spontaneous migration was measured with PBS plus 1% human serum albumin.
  • the upper well received ⁇ of whole blood from patients or controls diluted 1/10 in PBS.
  • the chambers were incubated for 3h at 37°C. Samples were stained with APC-anti- CD 15 (BD Biosciences) for 15 min, and 450 ⁇ 1 of lysis solution were then added. The total number of PMN added to the upper well and the number of PMN that migrated to the lower well were counted by flow cytometry using TruCount tubes (BD Biosciences).
  • NETs are composed of a DNA backbone decorated with granule proteins such as MPO, cit-H3 and elastase. NETs are also the major source of circulating cell free DNA. To identify NETs, we quantified serum NETs level by detecting MPO-DNA complexes in serum samples.
  • NETs from an healthy donor were produced and isolated, as previously described ⁇ Barrientos, 2014 #154 ⁇ . Briefly, freshly isolated PMNs were seeded in 12-well culture plates (1.5xl0 6 cells/well) and stimulated with 5 mM calciulm ionophore A23187 for 3 h at 37 °C with 5% C02. The cells were carefully washed twice with 1 ml PBS and then treated for 20 min at 37 °C with 20 U/ml restriction enzyme Alul in HBSS to recover large soluble NET fragments. Supematants were collected and centrifuged at 300 g for 5 min at 4 ⁇ C to remove contaminating cells and debris. NET preparations were then pooled, aliquoted, and stored at -80°C until use. DNA was quantified in NET samples by using PicoGreen (Molecular Probes), as previously described ⁇ Barrientos, 2014 #154 ⁇ .
  • NETs associated MPO-DNA complexes were then quantified as previously described ⁇ Kessenbrock, 2009 #155 ⁇ .
  • 5 ⁇ g/ml of mouse anti-human MPO antibody (ABD Serotec) was coated to 96-well microtiter plates. After blocking with 1% BSA, serum sample was added together with a peroxidase-labeled anti-DNA monoclonal antibody (component 2 of the Cell Death ELISA kit, Roche). After incubation, the peroxidase substrate was added according to the manufacturer's instructions. The optical absorbance was measured at 405 nm in an ELISA reader (Bio-Rad 550; Bio-Rad Laboratories, Tokyo, Japan). Samples were compared to the standard curve (from O. lng/ ⁇ to lOOng/ ⁇ ) and the results expressed in ng/ ⁇ .
  • Soluble cytokines (interleukin (IL)- 1 ⁇ , IL-6, TNF-a, G-CSF, IL-17, IL-18, IL-22, IL- 23, IL-10, TGF- ⁇ ), chemokines (IL-8, MCP-1), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metallo-proteinases (TIMP)-l were detected from plasma using Luminex - - assays (Luminex Performance Assays , R&D systems, Abingdon, UK); EDTA-whole-blood samples were centrifuged for 15 minutes at lOOOg within 30 minutes of collection. Assays were performed on crystored samples and diluted according to manufacturer's instructions (R&D systems).
  • Table 3 summarizes patients' clinical and AD-related characteristics. The baseline
  • MMSE of AD patients was 21 (range : 5-30) points. The group featured a slight imbalance with more females. As expected, there were significant differences across the diagnostic groups (MCI-AD and AD) for amyloid burden and cognitive deficits (MMSE and CDR). The prevalence of APOE e4 carriers was higher among patients with Alzheimer's disease. The level of C reactive protein levels was below 3 ⁇ g/ml in 54 subjects (18 controls and 36 patients) and did not exceed 10 ⁇ g/ml for both controls and patients.
  • Circulating PMNs from AD patients are highly activated and produce increased levels of ROS
  • PMN activation is associated with the modulation of surface molecules, specifically, a decrease in L-selectin (CD62L) or FCyRIIIB (CD 16b) and an increase in 2-integrins (CD l ib/CD 18, CDl lc/CD18) through either stimulus-induced shedding [Moldovan, 1999; Venturi, 2003] or translocation from intracellular granules [Sengelov, 1993].
  • Fig 1 A shows, we observed lower expression of CD62L and of CD 16b, associated with higher CD l ib and CDl lc expression, on resting PMNs from AD patients than from HC.
  • ROS production by PMNs from HC and AD patients was significantly increased compared to sample pre-incubated with PBS alone (Fig 1C).
  • ROS production was significantly higher in AD patients than in HC (Fig 1C) demonstrating the hypereactivity of PMNs to produce ROS in response to various stimuli.
  • ROS production by PMNs is a prerequisite for NETosis [Fuchs, 2007] .
  • PMN release neutrophil extracellular traps (NETs) that can entangle pathogens but also contribute to various inflammatory diseases.
  • NETs neutrophil extracellular traps
  • PMN are attracted by a variety of molecules, including formyl-methionyl-leucyl-phenyl-alaninc (fMLP), a bacterial peptide, and chemokines such as IL-8, which are released from sites of inflammation or injury.
  • fMLP formyl-methionyl-leucyl-phenyl-alaninc
  • chemokines such as IL-8
  • PMNs undergo phenotypic and functional changes from the time they are released into blood (fresh neutrophils) to the time they disappear from the circulation (aged neutrophils) in the absence of inflammation.
  • Aged or senescent CXCR4 hlgh PMNs are characterized by lower expression of CD62L, increased expression of ⁇ 2 integrins, as well as increased production of ROS and susceptibility of NET formation compared to the total circulating pool [Zhang, 2015] .
  • an immunosuppressive PMN subset CD16 bright /CD62L dim showing decreased adhesion properties and ROS production, has been reported during acute inflammation [Pillay, 2012] and healthy ageing [Sauce, 2017].
  • the ratio between the senescent and the immunosuppressive subsets was greater in AD-D patients compared to AD-MCI patients (Fig 3F).
  • ROS production by TLR4-primed PMNs from patients correlated with the percentages of the senescent CXCR4 high /CD62L low subset (Fig 3G) as well as with the percentage of the immunosuppressive PMN subset (Fig 3H). Similar results were observed after TLR1/2 or TNF priming and fMLP stimulation (not shown).
  • AD patients were dichotomized into slow decliners (SD) or fast decliners (FD) based on the progression of the CDR score at the last visit.
  • SD slow decliners
  • FD fast decliners
  • ROS production by PMNs from AD patients is associated with increased MMP-9 activity
  • Matrix metalloproteinase (MMP)-9 belong to a family of zinc containing endopeptidases that o degrade components of the extracellular matrix which is important for normal blood-brain barrier function [Yong, 2001]. Although circulating levels of MMP-9 were significantly lower in the two groups of AD patients than in HC, we found a decrease in serum levels of tissue inhibitors to the metalloproteinases (TIMP)-l (which inhibits preferentially MMP-9) [Brew, 2000] in AD-D patients compared to HC. This decrease leads the MMP- 9/TIMP-l ratio to increase. Of note, the MMP-9/TIMP-1 ratio correlated with the level of ROS produced by stimulated PMNs (Fig 8).
  • Serum level of IL-22 a bifunctional cytokine with both proinflammatory and protective functions, was significantly lower in the dementia AD group.
  • Serum level of IL-10 an anti-inflammatory cytokine, was significantly decreased in the AD dementia group compared to HC and the prodromal AD group.
  • Levels of other pro- (IL-23, G-CSF, MCP-1) and anti-inflammatory (TGFP) cytokines were similar in the two groups of AD patients and HC (Fig 7A1 , A2 and A3). No difference was observed for the different cytokine levels measured between both slow and fast decliner AD patients (not shown).
  • IL- 18 circulating level positively correlated with the percentage of the immunosuppressive PMN subset (data not shown) while IL-6 and IL-8 (circulating levels positively correlated with the percentage of the senescent PMN subset (data not shown) as well as with ratio between senescent and immunosuppressive PMN subsets (data not shown).
  • IL-6 and IL-8 circulating levels positively correlated with the percentage of the senescent PMN subset (data not shown) as well as with ratio between senescent and immunosuppressive PMN subsets (data not shown).
  • Alzheimer's disease met the following inclusion criteria: (i) progressive episodic memory impairment, characterized by low free recall not normalized by semantic cueing; (ii) absence of extrapyramidal signs; and (iii) a cerebrospinal fluid (CSF) profile of Alzheimer's disease, when available, defined as a score ⁇ 0.8, calculated with the formula amyloid-P42/[240 + (1.18 x T-tau)] .
  • CSF cerebrospinal fluid
  • MMSE Mini-Mental State Examination
  • CDR Clinical Dementia Rating
  • Alzheimer's disease Patients with Alzheimer's disease were classified in two groups according to disease severity, assessed by the CDR scale on the day blood samples were taken: sixteen patients had a score of 0.5, indicative of prodromal Alzheimer's disease with isolated episodic memory impairments and only moderate impact on the activities of daily living, and twenty-six patients had a score > 0.5, considered to demonstrate Alzheimer's disease dementia.
  • An extensive review was conducted of each patient's chart (Table 3). The two groups of patients were similar for age, prevalence of APOE ⁇ 4 carriers, duration and type of treatment, and levels of C reactive protein.
  • CBD corticobasal degeneration
  • PSP progressive supranuclear palsy
  • FTD frontotemporal dementia
  • n l l, aged 63-99 years (mean age, 74.2 years); MMSE : 13-28 (mean MMSE, 20.95)].
  • the differential diagnosis between non-Alzheimer's disease dementia and Alzheimer's disease was based on a non AD CSF biomarker profile defined by an Innotest Amylo ' id Tau Index > 1.2 (index IATI: ratio ⁇ - 42/(240 + 1,18 x tau).
  • CD62L, CD16b, CDl lb, CDl lc, CD83 and CD152 on resting neutrophils was studied on whole-blood samples maintained at 4°C and stained with specific mAbs (22).
  • samples kept on ice were incubated for 45 minutes with PE-Cy7-anti-human CXCR4 (clone 12G5, Sony Biotech, San Jose, CA), PE-anti-human CDl lb (clone ICRF44, BD Biosciences), and APC anti-human CD62L (BD Biosciences).
  • the immunosuppressive CD16bright/CD62Ldim neutrophil subset was studied after samples on ice were incubated for 45 minutes with FITC anti-human CD16 (clone 1D3, Beckman Coulter, Brea, CA), PE-anti-human CD1 lc (clone 3.9, Sony Biotech), PE-Cy7-anti-human CDl lb (clone Bear 1, Beckman Coulter), and APC anti- human CD62L (BD Biosciences). Samples were analyzed by flow cytometry.
  • Chemotaxis was assessed in Transwell plates (Corning Costar) containing 3- ⁇ pore polyvinylpyrrolidone-free polycarbonate filters.
  • the lower well of each chamber was filled with 600 ⁇ . of fMLP (10-7M) or of IL-8 (25 ng/niL) diluted in PBS plus 1% human serum albumin.
  • Spontaneous migration was evaluated with PBS containing 1 % human serum albumin.
  • the upper well was filled with 100 ⁇ , of whole blood from patients or controls diluted 1/10 in PBS. The chambers were incubated for 3 hours at 37°C and the migration index was evaluated as previously described.
  • NETs are composed of a DNA backbone peppered with granule proteins, including myeloperoxidase (MPO), cit-H3, and elastase.
  • MPO myeloperoxidase
  • NETs from a healthy donor were produced and isolated as previously described. Briefly, freshly isolated neutrophils were seeded in 12-well culture plates (1.5x 106 cells/well) and stimulated with 5 mM calcium ionophore A23187 for 3 hours at 37 °C with 5% C02. The cells were carefully washed twice with 1 mL PBS and then treated for 20 minutes at 37 °C with 20 U/mL restriction enzyme Alul in HBSS to recover large soluble NET fragments.
  • MPO myeloperoxidase
  • ABTS peroxidase substrate
  • Optical absorbance was measured at 405 nm in an ELISA reader (Bio-Rad 550; Bio-Rad Laboratories, Tokyo, Japan). Samples were compared to the standard curve (from 0.1 ng/ ⁇ to 100 ng/ ⁇ ), and the results expressed in ng/ ⁇ .
  • Soluble cytokines, chemokines, MMP-9, and TIMP-1 were detected from serum with Luminex assays (Luminex Performance AssaysTM, R&D Systems). Whole-blood samples were centrifuged for 15 minutes at 1000 g within 30 minutes of collection. Assays were performed on cryostored samples frozen at -80°C and diluted according to manufacturer's instructions (R&D Systems).
  • neutrophils were further purified by negative selection with pan anti-human HLA class II-coated magnetic beads (Miltenyi Biotec, CA) to deplete B lymphocytes, activated T lymphocytes and monocytes, as previously described. Less than 1% of cells were positive by non specific esterase staining, and flow cytometry showed the absence of CD45+/CD14high, CD45+/CD3+ or CD45+/CD19+ cells; this showed that the neutrophils were highly purified, without contaminating monocytes.
  • pan anti-human HLA class II-coated magnetic beads Miltenyi Biotec, CA
  • Isolated neutrophils (5xl06/ml) were cultured for 24 h at 37°C with 5% C02 in 24-well tissue culture plates (Costar) in RPMI 1640 medium (Sigma- Aldrich) in the presence of PBS Tumor necrosis factor (TNF)-a, or the following Toll-Like Receptor (TLR) agonists: Pam3CSK4 (TLR1/2 agonist, 10 ⁇ g/mL), or LPS (TLR4 agonist, 100 ng/mL).
  • TNF Tumor necrosis factor
  • TLR Toll-Like Receptor
  • Supernatants were stored at -80°C for no longer than 15 days before assay.
  • IL-8, IL-6, IL- ⁇ , TNFa, and IL- 17 were detected simultaneously in supernatants with Luminex assays.
  • RNA libraries were prepared with 500 nanograms of total RNA from each individual using the TruSeq Stranded Total RNA (Illumina, San Diego, CA) according to the manufacturer's instructions. Libraries were analyzed on a Tapestation (Agilent Technologies, Les Ulis, France) - - and then sequenced on a NextSeq500 (Illumina) as 75-bp paired-end reads with a sequencing depth of 30 million reads.
  • Circulating neutrophils from the Alzheimer disease dementia group are highly activated and produce higher levels of ROS and intravascular NETs than those from healthy subjects - -
  • AD-MCI prodromal Alzheimer's disease group
  • AD-D Alzheimer's disease dementia group
  • neutrophils Upon activation, neutrophils are reported to trigger microbicidal mechanisms, such as ROS production and release of NETs.
  • ROS production was significantly higher in unstimulated neutrophils from AD-D patients than HC (Data not shown).
  • ROS production did not differ significantly between MCI- AD patients and HC.
  • ROS production by non-primed neutrophils was significantly higher in AD-D patients than in either HC or AD-MCI patients (Data not shown).
  • Neutrophil chemotaxis towards IL-8 or fMLP was significantly lower in AD-D patients than in either HC or AD-MCI patients (Data not shown). Similar results was observed in supplemental experiments performed on isolated neutrophils (Data not shown) ruling out the influence of dendritic cells and monocytes derived cytokines and chemokines on chemotaxis of whole-blood neutrophils. As previously reported, we observed a significantly lower phagocytic index in patients with Alzheimer's disease (Fig Data not shown), perhaps related, at least, in part to the decreased CD 16b (FcyRIII) expression on neutrophil surfaces (Data not shown). Neutrophil phagocytic activity was also significantly lower in the samples from AD-D than AD-MCI patients.
  • the percentage of neutrophils in the CD 16bright/ CD62Ldim immunosuppressive subset was higher in AD-MCI patients than in HC, but significantly lower in AD-D patients than in HC (Data not shown). Accordingly, the ratio between the senescent and immunosuppressive subsets was higher in AD-D than in AD-MCI patients (Data not shown).
  • Phenotypic changes associated with neutrophil activation involve both molecular rearrangements to change the activity and/or sub-cellular localisation of pre-existing molecules. - -
  • Activation also includes increased ability of neutrophils to synthesize de novo proteins such as cytokines and chemokines, although neutrophils possess 10-20 times less RNA than other leukocytes.
  • Transcriptome sequencing (RNA-Seq) was carried out on mRNA purified from freshly isolated neutrophils from healthy controls and patients with Alzheimer's disease. Analysis of RNA isolated from unstimulated neutrophils revealed expression (CPM >20) of 12289 known genes among a total of 22675 genes analyzed.
  • actin cytoskeleton is required for vesicle trafficking to the plasma membrane and granule exocytosis allowing the NADPH oxidase cytosolic components to translocate to the membrane and to form the catalytically active enzyme complex.
  • Functions associated with cell senescence and death, various signaling pathways, nucleic acid metabolism, lipid and protein metabolism, regulators of protein synthesis or stability including ubiquitination machinery and chaperones were similarly upregulated (Data not shown).
  • Neutrophils from AD-D patients showed significant upregulation (p ⁇ 0.05) of 18 transcripts including inflammatory and host defense genes (TAP1, CD83, CTLA4, TMIGD2, S100B, EDAR, MDS2) (Data not shown).
  • CD83 encodes an Ig supergene family member, best known as a marker for mature dendritic cells, but has also been identified as a differentially regulated gene in neutrophil-transcription profiles.
  • CD83 surface antigen has been reported previously in neutrophils cultured in vitro with cytokines and bacterial peptides or during acute bacterial infection and chronic inflammatory disease in vivo.
  • Increased expression of CD83 transcript - - was also associated with up-regulation of TAP 1, involved in the proteolytic steps of MHC-I antigen processing by neutrophils suggesting that neutrophils from AD-D patients can adapt a function as antigen presenting cell.
  • CTLA4 encodes Cytotoxic T-Lymphocyte Associated Protein 4 (CD 152) which transmits an inhibitory signal to T cells and is also involved in neutrophil regulation.
  • CD 152 Cytotoxic T-Lymphocyte Associated Protein 4
  • SIK1 a serine/threonine kinase, that belongs to the AMP activated protein kinase family
  • CRIP2 that belongs to the cysteine-rich intestine protein family 1 act as transcriptional repressors of NF-KB-mediated transcription induced by neutrophil in response to pro-inflammatory mediators such as TLR agonists.
  • AK5 is an adenylate kinase that catalyzes the reversible transfer of the ⁇ -phosphate group from a phosphate donor (normally ATP) to AMP, and generate the intracellular second messenger cAMP.
  • cAMP significantly inhibits ROS production by neutrophils in young subjects but increases ROS level in the 50-80 years old individuals.
  • GRASP promotes activation of ADP-ribosylation factor-6 (Arf6), a low molecular weight GTPase that regulates key aspects of endocytic recycling pathways and that has been involved in neutrophil degranulation and oxidative burst.
  • ADP-ribosylation factor-6 Arf6
  • DTX1, PSMB8 ubiquitination machinery
  • NETosis NETosis in these patients.
  • PERI encodes for period circadian protein homo log 1 protein, a key regulator of internal biological clock in neutrophils, which has been involved in the mobilization of young neutrophils from the bone marrow and their ageing in the peripheral circulation.
  • neutrophil markers were similarly associated with the overall disease progression profile over a two-year follow-up period, determined by retrospectively taking into account the clinical scores one year before baseline. We observed that both the neutrophil activation parameters and the percentage of the different neutrophil subpopulations were correlated with AMMSE and ACDR-SOB (Data not shown).
  • ROS production by peripheral neutrophils from patients with Alzheimer's disease correlates with brain amyloid burden
  • MMP-9 matrix metalloproteinase
  • BBB blood-brain barrier
  • Active MMP-9 forms 1 : 1 complexes with tissue inhibitor of metalloproteinases-1 (TIMP-1), which preferentially inhibits it, and the balance between them reflects the net proteolytic activity in several physiological processes.
  • TIMP- 1 tissue inhibitor of metalloproteinases-1
  • the MMP-9 and TIMP- 1 levels were significantly lower in both groups of patients than in HC (Data not shown). This led to a higher MMP-9/TIMP-1 ratio, which was moreover significantly different in ADD patients than in HC (Data not shown).
  • the MMP-9/TIMP-1 ratio also correlated with the level of ROS produced by unstimulated and fMLP-stimulated neutrophils (Data not shown).
  • Pro-inflammatory cytokines have been reported to be overproduced by PBMCs from patients with Alzheimer's disease compared to healthy controls. As neutrophil activity is highly regulated by proinflammatory mediators such as cytokines, we thus investigated the peripheral cytokine environment in patients. Serum levels of the proinflammatory cytokines Interleukin (IL)-ip, IL-6, TNFa, and IL-18 were significantly higher in both groups of patients than in HC (Data not shown). Serum levels of IL-8 and IL-17 were also significantly higher in the AD-D patients than in HC (Data not shown) and negatively correlated with the MMSE score (Data not shown).
  • IL Interleukin
  • the serum level of IL-22 was significantly lower in the AD-D group than in HC.
  • the serum level of IL-10 was significantly lower in the AD-D group than in either HC or AD-MCI patients (Data not shown).
  • HMGB1 a damage-associated molecular pattern molecule, on polymorphonuclear neutrophil migration depends on its concentration. J Innate Immun 4:41-58.
  • beta-amyloid activates the 0-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease. J Biol Chem 274:15493-15499.
  • Cattaneo A., N. Cattane, S. Galluzzi, S. Provasi, N. Lopizzo, C. Festari, C. Ferrari, U.P. Guerra, B. Paghera, C. Muscio, A. Bianchetti, G.D. Volta, M. Turla, M.S. Cotelli, M. Gennuso, A. Prelle, O. Zanetti, G. Lussignoli, D. Mirabile, D. Bellandi, S. Gentile, G. Belotti, D. Villani, T. Harach, T. Bolmont, A. Padovani, M. Boccardi, G.B. Frisoni, and I.-F. Group. 2017. Association of brain amyloidosis with pro-inflammatory gut bacterial taxa and peripheral inflammation markers in cognitively impaired elderly. Neurobiol Aging 49:60-68.
  • Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia. Am J Physiol Heart Circ Physiol 289:H558-568.
  • Anti-myeloperoxidase antibodies enhance phagocytosis, IL-8 production, and glucose uptake of polymorphonuclear neutrophils rather than anti-proteinase 3 antibodies leading to activation-induced cell death of the neutrophils.
  • Amnestic syndrome of the medial temporal type identifies prodromal AD: a longitudinal study. Neurology 69: 1859-1867.

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Abstract

La présente invention concerne la maladie d'Alzheimer et le procédé pour déterminer sa progression et sa gravité. Les inventeurs ont géré une étude pour caractériser phénotypiquement et caractériser fonctionnellement des PMN circulants humains dans le sang total à différents stades de maladie chez des patients atteints d'Alzheimer. Ils ont également étudié des associations entre ces indicateurs et le taux de progression de la maladie. Ils ont également montré (par dosages Luminex) que certaines protéines et en particulier certaines cytokines pourraient être utilisées pour prédire la gravité de la maladie d'Alzheimer. Ainsi, la présente invention concerne des procédés de prédiction de la rapidité de progression de la maladie d'Alzheimer ou de la gravité de la maladie chez un patient par détermination de l'état d'activation de neutrophiles ou du niveau d'expression de différentes protéines (en particulier des cytokines).
PCT/EP2018/057511 2017-03-24 2018-03-23 Procédé de prédiction de la progression de la maladie d'alzheimer WO2018172540A1 (fr)

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Cited By (3)

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CN109342738A (zh) * 2018-11-19 2019-02-15 深圳大学 一组血清差异蛋白组合在制备用于检测阿尔茨海默症的试剂中的应用
US20220093272A1 (en) * 2018-12-06 2022-03-24 B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University Integrated system and method for personalized stratification and prediction of neurodegenerative disease
WO2025021053A1 (fr) * 2023-07-21 2025-01-30 中南大学 Réactif pour la détection des neutrophiles vieillissants dans le sang et son utilisation

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CN109342738B (zh) * 2018-11-19 2022-04-15 深圳大学 一组血清差异蛋白组合在制备用于检测阿尔茨海默症的试剂中的应用
US20220093272A1 (en) * 2018-12-06 2022-03-24 B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University Integrated system and method for personalized stratification and prediction of neurodegenerative disease
US11915833B2 (en) * 2018-12-06 2024-02-27 B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University Integrated system and method for personalized stratification and prediction of neurodegenerative disease
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