ALZHEIMER'S DISEASE EARLY DIAGNOSIS AND/OR PROGNOSIS IN CIRCULATING IMMUNE CELLS BASED ON HEPARAN SULFATES AND/OR OF HEPARAN SULFATE SULFOTRANSFERASES

Abstract

The present invention relates to a method of prognosis and/or diagnosis of Alzheimer's disease by determining the level and/or cellular distribution of heparan sulfates (HS) and/or heparan sulfate sulfotransferases (HSSTs) from isolating circulating immune cells in said circulating immune cells.

Claims

1. An in vitro method of prognosis and/or diagnosis of Alzheimer's disease, in a subject comprising the steps of: a) isolating circulating immune cells from said subject; b) determining the level and/or cellular distribution of heparan sulfates (HS) and/or heparan sulfate sulfotransferases (HSSTs) in said circulating immune cells; c) comparing: said level and/or cellular distribution of HS and/or HSSTs; or the ratio of said level of HSSTs to said level of HS, to a respective reference representing a known disease or health status.

2. The in vitro method according to claim 1, wherein said HS is 3-O-sulfated heparan sulfates (3S-HS), and said HSSTs are selected from heparan sulfate 3-O-sulfotransferases (HS3ST), preferentially HS3ST1, HS3ST2, HS3ST3, HS3ST4, HS3ST5 and/or HS3ST6, more preferentially HS3ST2, HS3ST3A and/or HS3ST3B.

3. The in vitro method according to claim 1, wherein said circulating immune cells are circulating T cells, B cells, monocytes and/or monocyte derived macrophages (MDM), preferentially circulating monocytes and/or MDM.

4. The in vitro method according to claim 1, wherein said method further comprises after said step a) a culturing step a1) of said circulating immune cells in an appropriate culture medium, such as RPMI 1640 medium.

5. The in vitro method according to claim 4, wherein said culturing step a1) is performed between 7-10 days, preferably 10 days, within said appropriate culture medium complemented with M-CSF until said monocytes and/or MDM present a M0 phenotype.

6. The in vitro method according to claim 5, wherein said culturing step, following said step a1), comprises an additional culturing step a2) of 2 to 3 days, preferably 3 days, in said appropriate culture medium complemented with M-CSF which further comprises anti-inflammatory factors, preferably IL4/IL10, until said M0 macrophage presents a M2 macrophage phenotype, or which further comprises pro-inflammatory factors, preferably Toll-like receptor (TLR) ligands, LPS and IFN?, until said M0 macrophage presents a M1 macrophage phenotype.

7. The in vitro method according to claim 1, wherein said level of anyone of HS and HSSTs is determined by a method selected from Immunofluorescence, Western Blot, ELISA, mass spectrometry, flow cytometry methods, immunohistochemistry methods, and combination thereof.

8. The in vitro method according to claim 1, wherein said level and/or said cellular location of HS3ST is altered.

9. The in vitro method according to claim 1, wherein said level of HS or 3S-HS is altered and/or its cellular location is altered to be accumulated essentially into the cytosol.

10. A circulating biological marker for Alzheimer's disease consisting of at least one of HS and/or HSSTs, preferably 3S-HS and/or HS3ST, said HS3ST being preferentially selected from HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5 and/or HS3ST6, preferably HS3ST2, HS3ST3A, HS3ST3B and/or HS3ST5, for use as a circulating biological marker for Alzheimer disease.

11. A kit for the prognosis and/or diagnosis of Alzheimer's disease comprising purification means of circulating immune cells, preferably monocytes and/or monocytes derived macrophages (MDM), and detection means of level and/or cellular distribution of HS and/or HSSTs, preferably 3S-HS and/or HS3ST, said HS3ST being preferentially selected from HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5 and/or HS3ST6, preferably HS3ST2, HS3ST3A, HS3ST3B and/or HS3ST5.

12. A kit according to claim 11 comprising: at least one primer combination to amplify said HS and/or HSSTs, preferably 3S-HS and/or HS3ST, said HS3ST being preferentially selected from HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5 and/or HS3ST6, preferably HS3ST2, HS3ST3A, HS3ST3B and/or HS3ST5; and/or at least one probe, such as nucleic acid probes, to detect said HS and/or HSSTs, preferably 3S-HS and/or HS3ST, said HS3ST being preferentially selected from HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5 and/or HS3ST6, preferably HS3ST2, HS3ST3A, HS3ST3B and/or HS3ST5; and/or at least one specific antibody of said HS and/or HSSTs, preferably 3S-HS and/or HS3ST, said HS3ST being preferentially selected from HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5 and/or HS3ST6, preferably HS3ST2, HS3ST3A, HS3ST3B and/or HS3ST5.

13. A kit according to claim 12 wherein said specific antibody is a conjugated antibody linked to a colorimetric or fluorescent label.

14. A kit according to claim 11, wherein said purification means comprise filters selectively retaining circulating immune cells, preferably circulating monocytes and/or monocytes derived macrophages (MDM).

15. A kit according to claim 11, wherein said kit further comprises: Buffers, preferably Phosphate Buffered Saline; an appropriate cell culture medium, such as RPMI 1640 medium; M-CSF; optionally pro and/or anti-inflammatory factors, preferably selected from recombinant human cytokines IL4 and IL10; or Toll-like receptor (TLR) ligands, LPS and IFN?; and one or more cell culture containers.

Description

FIGURES

[0121] The invention will be further described and illustrated with reference to the accompanying drawings in which:

[0122] FIG. 1. Method of isolating Monocytes (CD14+ cells) from a blood sample. PMBC are isolated from EDTA blood samples by methods classically used by the men of the art, for instance Ficoll devises, magnetic beads or cell selecting filters. Monocytes are further isolated from PBMC by strategies classically used by the men of the art, for instance using CD14-magnetic beads. Obtained monocytes (CD14+) can be used for direct analysis or for primary culture.

[0123] FIG. 2. Method of cultured isolated monocytes (CD14+) and differentiation to obtain M0, M1 or M2 macrophage phenotypes. Culture conditions are as those used by the men of the art. For instance, monocytes are cultured in RPMI 1640 containing 10% HuS. Adherent cells are then cultured in the presence of M-CSF to obtain M0 phenotype. M1 phenotype is obtained by including pro-inflammatory factors, for instance IFN? and LPS, in the cell medium of M0 cultured cells. M2 phenotype is obtained by including anti-inflammatory factors, for instance IL4 and IL10, in the cell medium of M0 cultured cells. Compounds concentrations and culturing times are as those showed in the figures.

[0124] FIG. 3. Accumulation and cellular distribution of 3S-HS and HS3ST2 in MDM M0 from Patient 1 and Control individual 1. Differential cellular accumulation and distribution of 3S-HS and HS3ST2 immunostaining in MDM M0 from an AD patient vs control individual. Cell nuclei is shown by DAPI staining. A) Confocal optical slide at a z plane near the cell basement, B) confocal optical slide at a z plane at the middle cell level, C) total confocal projection of the entire cell, and D) phase contrast.

[0125] FIG. 4. Accumulation and cellular distribution of 3S-HS and HS3ST2 in MDM M0 from AD Patient and Control individual. Differential cellular accumulation and distribution of 3S-HS and HS3ST2 immunostaining in MDM M0 from AD vs control individuals. Cell nuclei is shown by DAPI staining. A) Confocal optical slide at a z plane near the cell basement, B) confocal optical slide at a z plane at the middle cell level, C) total confocal projection of the entire cell, and D) phase contrast.

[0126] FIG. 5. Accumulation of 3S-HS in MDM M0 from Alzheimer's disease vs inflammatory disease Osteoarthritis (OA). The figure shows 5 Alzheimer's disease (AD) patients, 5 control individuals and 5 Osteoarthritis (OA) patients. Total projections of the MDM M0 cells from AD cells shows characteristic AD immunostaining

EXAMPLES

Material and Methods

Isolation of Circulating Immune Cells

[0127] Peripheral Blood Mononuclear Cells (PBMC) isolation. K2-EDTA venous blood samples are used to isolate PBMC. Ficoll method is used for separating and isolation PBMCmost specifically lymphocytes and monocytes. Blood specimens are carefully layered on top of the Ficoll-Paque Plus solution, and then briefly centrifuged to form different layers containing different types of cells. The bottom layer is made up of red blood cells (erythrocytes) which are collected or aggregated by the Ficoll medium and sink completely through to the bottom. The next layer up from the bottom is primarily granulocytes, which also migrate down through the Ficoll-Paque Plus solution. The next layer toward to top, which is typically at the interface between the plasma and the Ficoll solution, is the lymphocytes along with monocytes and platelets. To recover these cells, Ficoll-Paque Plus fabricant instructions are followed, this is largely known by men or the art.

[0128] Lymphocytes and monocytes independent isolation and characterization. After Ficoll isolation of PBMC, lymphocytes and monocytes are independently isolated by using immune-magnetic beads (Myltenyi MACS microbeads) on the basis of surface markers selections with monoclonal specific antibodies. T cells are separated by using CD3+ coated beads. B cells are separated by using CD19+ coated beads. Monocytes are separated by using CD14+ coated beads. This technique, well known by the skill person, allows the separation of cells in relatively short time. Cells purity is assessed by FACScan analysis with the specific antibodies as classically performed by the skilled person. T cells are CD3+, B cells are CD19+, monocytes are CD14+ and CD68+. Cell purity is typically of at least 95% when assessed by flow cytometry. Each experiment is conducted with cells isolated from a single donor, in any case cells from different donors are combined.

Culture and Differentiation of Macrophage to M0, M1 and M2 Phenotype (FIG. 1)

[0129] MDM phenotypes M0, M1 and M2 induction. Freshly isolated monocytes (CD14+ cells) are cultured in complete RPMI 1640 medium (Gibco 21875-034) containing 10% human serum (HuS, male), classically at 0.2/1?10.sup.6 cells/cm.sup.2. Cells are stimulated for instance during 7-10 days with M-CSF (for instance 50 ng/mL) to induce MDM to M0 phenotype; then cells are further stimulated for instance during 2-3 days with M-CSF (for instance 50 ng/mL) with inflammatory cytokines or with Toll-like receptor (TLR) ligands (for instance LPS 10 ng/mL; IFN? 50 ng/mL) to induce the polarization of M0 macrophage into M1 phenotype or with M-CSF (for instance 50 ng/mL) and recombinant human anti-inflammatory cytokines (for instance IL4 20 ng/mL; IL10 20 ng/mL) to induce the polarization of M0 macrophage into M2 phenotype. MDM M0 is characterized by flow cytometry as CD14+, CD68+, CD80?, CD163?, CD206?, and CD209?. MDM M1 is characterized by flow cytometry as CD14+, CD68+, CD80+, CD163?, CD206?, and CD209?. MDM M2 is characterized by flow cytometry as CD14+, CD68+, CD80?, CD163+, CD206+, and CD209+.

[0130] Immunofluorescence. Cultured PBMC whatever the phenotype is (T cells, B cells, monocytes, or MDM M1, M2, or M0) are separately labelled with specific antibodies. Heparan sulfates in cells are labelled with HS-recognizing antibody HS4C3 (3S-HS), which is a phage display antibody able to selectively detect HS-3S, or by any other antibodies able to selectively detect HS having 3-O-sulfates), or by any other anti-HS antibody, and by 3-O-sulfotransferases (HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5, or HS3ST6) recognizing antibodies. Labelling is revealed as classically do by the skilled person by using the secondary and tertiary antibodies coupled to fluorescent probes (Alexa-Fluor probes are here classically used). The phage display HS4C3 antibody was revealed by an anti-VSV antibody and the by a fluorescent tertiary antibody.

[0131] Cell morphology, HS and HS3ST levels and cellular localization are assessed by microscopy, flow cytometry, fluorescence microscopy and/or by confocal fluorescence microscopy.

[0132] Other detection techniques such as Western blot, ELISA or other immunologic techniques can also be used.

Example 1: HS3ST2 and HS-3S Immunolabelling in M2 MDM from AD and Control Individuals

[0133] Blood was collected from a control and an AD subject. AD patient was clinically characterized and previously diagnosed to be AD by amyloid uptake as measured by Pittsburg compound B positron emission tomography (PiB-PET) as do the men of the art. PBMC were isolated as described in materials and methods as do the men of the art. Isolated PBMC were used to isolate monocytes (CD14.sup.+ cells) that were then cultured in complete RPMI 1640 medium (Gibco 21875-034) containing 10% HuS (male) at 0.2/1?10.sup.6 cells/cm.sup.2. Cells were then stimulated during 10 days with M-CSF (50 ng/mL) to induce MDM to M0 phenotype. M0 MDM were characterized by flow cytometry with next antibodies CD14+, CD68+, CD80?, CD163?, CD206?, and CD209?, as classically do the men of the art. Cultured MDM M0 were then labelled with specific primary antibodies against HS3ST2 and HS4C3. HS3ST2 labelling was revealed as classically do a skilled person by using the secondary antibody labelled with Alexa-Fluor 555. The phage display HS4C3 antibody was revealed by an anti-VSV antibody and then by a tertiary antibody labelled with Alexa-Fluor 488. Stack images were obtained with the software CellSens from a spinning disk inverted confocal microscope (IX81 DSU Olympus, 60N.A. 1.35) coupled to an Orca Hamamatsu RCCD camera. Images were processed with the ImageJ software (W. Rasband, National Institute of Health).

Results

[0134] FIG. 3 shows the altered cellular accumulation and altered distribution of 3S-HS and HS3ST2 in the cell membrane and at the intracellular level of MDM M0 from AD vs control individuals. 3S-HS enhanced immunostaining in AD patient is observed in both confocal optical slides at a z axis plane near the cell basement (FIG. 3A1), at the middle of the cell (FIG. 3B1), and reflected by the total confocal projection of the entire cell (FIG. 3C1). HS3ST2 immunostaining shows altered cellular accumulation, while in control cells HS3ST2 accumulates in the ruffling regions near the cell basement (FIG. 3A2), in AD HS3ST2 acquired a diffuse cytosolic distribution (FIG. 3B2). This intracellular cellular relocation 3S-HS is also observed in the total confocal projection of the entire cell (FIG. 3C2).

Conclusion

[0135] Compared to control MDM M0, AD shows morphologically altered MDM M0, which accumulate 3S-HS at the intracellular level and at the cell membrane. This is accompanied by an altered cell distribution of HS3ST2, which disappears from the ruffing areas to be located at the cytosol in the AD cells. In conclusion, the differential 3S-HS levels and distribution observed in AD can be used to determine whether a patient is affected or not by AD.

Example 2: HS3ST2 and HS-3S Immunolabelling in M0 MDM from AD and Control Individuals

[0136] Blood was collected from a second control and a second AD subject. AD patient was clinically characterized and previously diagnosed to be AD by amyloid uptake as measured by Pittsburg compound B positron emission tomography (PiB-PET) as do the men of the art. PBMC were isolated as described in materials and methods as do the men of the art. Isolated PBMC were used to isolate monocytes (CD14.sup.+ cells) that were then cultured at 1?10.sup.6 per mL in complete RPMI 1640 medium (Gibco 21875-034) containing 10% HuS (male) at 0.2/1?10.sup.6 cells/cm.sup.2. Cells were then stimulated during 7 days with M-CSF (50 ng/mL) to induce MDM to M0 phenotype. M0 MDM were characterized by flow cytometry with next antibodies CD14+, CD68+, CD80?, CD163?, CD206?, and CD209?, as classically do the men of the art. Cultured MDM M0 were then labelled with primary antibodies HS3ST2 and HS4C3. HS3ST2 labelling was revealed as classically do a skilled person by using the secondary antibody labelled with Alexa-Fluor 555. The phage display HS4C3 antibody was revealed by an anti-VSV antibody and then by a tertiary antibody labelled with Alexa-Fluor 488. Stack images were obtained with the software CellSens from a spinning disk inverted confocal microscope (IX81 DSU Olympus, 60N.A. 1.35) coupled to an Orca Hamamatsu RCCD camera. Images were processed with the ImageJ software (W. Rasband, National Institute of Health).

Results

[0137] FIG. 4 shows the altered cellular accumulation and altered distribution of 3S-HS and HS3ST2 in the cell membrane and at the intracellular Level of MDM M0 from AD vs control individuals. 3S-HS enhanced immunostaining in AD patient is observed in both confocal optical slides at a z axis plane near the cell basement (FIG. 4A1), at the middle of the cell (FIG. 4B1), and reflected by the total confocal projection of the entire cell (FIG. 3C1). HS3ST2 immunostaining shows altered cellular accumulation, while in control cells HS3ST2 accumulates in the ruffling regions near the cell basement (FIG. 4A2), in AD HS3ST2 acquired a diffuse cytosolic distribution (FIG. 4B2). This intracellular cellular relocation 3S-HS is also observed in the total confocal projection of the entire cell (FIG. 4C2).

Conclusion

[0138] Compared to control MDM M0, AD shows morphologically altered MDM M0, which accumulate 3S-HS at the intracellular level and at the cell membrane. This is accompanied by an altered cell distribution of HS3ST2, which disappears from the ruffing areas to be located at the cytosol in the AD cells. In conclusion, the differential 3S-HS levels and distribution observed in AD can be used to determine whether a patient is affected or not by AD.

Example 3: 3S-HS Immunostaining of 5 AD Patients vs 5 Control Individuals and 5 Patients Affected by Osteoarthritis (OA) and Neurologically Normal (any Detectable Dementia)

[0139] Age of the OA Patients Chosen to Match with Age of the AD Patients

[0140] Blood from 5 different AD patients and 5 control individuals and 5 neurologically normal OA patients (all of similar ages) was collected and processed to obtain MDM M0 cells, which were immunostained for 3S-HS as in example 1. Stack images were obtained with the software CellSens from a spinning disk inverted confocal microscope (IX81 DSU Olympus, 60N.A. 1.35) coupled to an Orca Hamamatsu RCCD camera. Images were processed with the ImageJ software (W. Rasband, National Institute of Health).

Results

[0141] Cellular accumulation and altered distribution of 3S-HS were analyzed in MDM M0 from 5 different AD patients and 5 control individuals and 5 neurologically normal OA patients (all of similar ages). FIG. 5 total confocal projections of the entire 3S-HS immunostained cells. 3S-HS accumulated at the intracellular level and in the cell membrane of MDM M0 from AD compared to control individuals and OA and control individuals, which did not show the AD phenotype.

Conclusion

[0142] Compared to control and OA MDM M2 from control individuals, MDM M2 from AD shows morphologically altered cells which accumulate 3S-HS at the intracellular level and at the cell membrane. This is accompanied by an altered cell distribution of HS3ST2, which disappears from the ruffing areas to be located at the cytosol in the AD cells. In conclusion, the differential 3S-HS levels and distribution observed in AD can be used to determine whether a patient is affected or not by AD. Results from OA patients indicates that the observed AD phenotype is not related to an inflammatory related condition.

Example 4: Cell Diameter, Area and 3S-HS and HS3ST2 Immunostaining in 5 AD Patients vs 5 Control Individuals

[0143] Blood from 5 different AD patients and 5 control individuals was collected and processed to obtain MDM M0 cells, which were immunostained for 3S-HS as in example 1. Stack images were obtained with the software CellSens from a spinning disk inverted confocal microscope (IX81 DSU Olympus, 60N.A. 1.35) coupled to an Orca Hamamatsu RCCD camera.

[0144] Images were processed with the ImageJ software (W. Rasband, National Institute of Health) to obtain cell diameter (as classically do the man of the art) for an average of 50 cells for each AD patient or each control subject observed at different fields. The fields were aleatory selected. Results are shown in Table 1 (n=5). Table 1 also shows the 2D interaction surface calculated as the average area of 50 cells observed in the 2D images. This area, expressed in ?m.sup.2, was calculated with the ImageJ software as classically do by the person skilled in the art. This represents the average area calculated on 50 cells from each AD-patient (n=5) or control subject (n=5). Fluorescence intensities, expressed in arbitrary units (AU), for 3S-HS and HS3ST2 were obtained by the ImageJ software from the analysis of 50 cells for each AD patient (n=5) or each control subject (n=5). The fluorescence was measured in the areas of the cell concentrating the concentrated fluorescence intensity. The fields were aleatory taken (Table 1). Results were similar and consistent in the different observed fields.

Results

[0145] Table 1 shows that MDM M0 from Alzheimer's disease patient have increased size as shown by the increased diameter and 2D interaction surface (Table 1). 3S-HS fluorescent intensity was 3 times higher in MDM M0 from Alzheimer's disease that that measured in control individuals. HS3ST2 fluorescent intensity was twice lower. The ration of the 3S-HS/HS3ST2 fluorescence shows a significate difference between AD vs control individuals. This ratio is 6 times higher in Alzheimer disease MDM M0.

TABLE-US-00001 TABLE 1 MDM M0 size, 2D surface interaction, and levels of fluorescence intensity of preferential location sites for 3S-HS and HS3ST2 in AD patients and control individuals. 2D Interaction 3S-HS/HS3ST2 Diameter surface 3S-HS HS3ST2 ratio ?m ?m.sup.2 Fluorescence relative intensity (AU) Control patients 45.91 +/? 6.196 1683.93 +/? 453.33 1305.90 +/? 448.04 1147.07 +/? 327.378 1.14 AD patients 76.46 +/? 9.82 4664.52 +/? 1194.62 3917.68 +/? 944.12 613.13 +/? 112.529 6.39

Conclusion

[0146] Results in Table 1 shows that cell diameter, 2D interaction surface, 3S-H, HS3ST2, and 3S-HS/HS3ST2 can be used to identify Alzheimer disease in MDM M0 cells. This opens to the use of any of these parameters or a combination of them in the diagnostic or prognostic of Alzheimer's disease.

REFERENCES

[0147] Anoop A, Singh P K, Jacob R S, Maji S K. CSF Biomarkers for Alzheimer's Disease Diagnosis. Int J Alzheimers Dis. 2010; 2010.

[0148] Baird A L, Westwood S, Lovestone S. Blood-Based Proteomic Biomarkers of Alzheimer's Disease Pathology. Front Neurol. 2015; 6:236.

[0149] Ballard C, Gauthier S, Corbett A, Brayne C, Aarsland D, Jones E. Alzheimer's disease. Lancet 2011; 377 (9770):1019-31.

[0150] Boss? P, Spalletta G, Caltagirone C, Ciaramella A. Myeloid Dendritic Cells are Potential Players in Human Neurodegenerative Diseases. Front Immunol. 2015; 6:632.

[0151] Cavedo E, Lista S, Khachaturian Z, Aisen P, Amouyel P, Herholz K, Jack C R Jr, Sperling R, Cummings J, Blennow K, O'Bryant S, Frisoni G B, Khachaturian A, Kivipelto M, Klunk W, Broich K, Andrieu S, de Schotten M T, Mangin J F, Lammertsma A A, Johnson K, Teipel S, Drzezga A, Bokde A, Colliot O, Bakardjian H, Zetterberg H, Dubois B, Vellas B, Schneider L S, Hampel H. The Road Ahead to Cure Alzheimer's Disease: Development of Biological Markers and Neuroimaging Methods for Prevention Trials Across all Stages and Target Populations J Prey Alzheimers Dis. 2014; 1 (3):181-202;

[0152] Fiala M, Veerhuis R. Biomarkers of inflammation and amyloid-beta phagocytosis in patients at risk of Alzheimer disease. Exp Gerontol. 2010; 45 (1):57-63).

[0153] Goedert M, Jakes R, Spillantini M G, Hasegawa M, Smith M J, et al. Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans. Nature 1996; 383: 550-3.

[0154] Gong C X, Iqbal K. Hyperphosphorylation of microtubule-associated protein tau: a promising therapeutic target for Alzheimer disease. Curr Med Chem. 2008; 15 (23):2321-8.

[0155] Hernandez F, Perez M, Lucas J J, Avila J. Sulfo-glycosaminoglycan content affects PHF-tau solubility and allows the identification of different types of PHFs. Brain Res. 2002; 935: 65-72.

[0156] Iqbal K, Liu F, Gong C X, Alonso Adel C, Grundke-Iqbal I. Mechanisms of tau-induced neurodegeneration. Acta Neuropathol. 2009; 118 (1):53-69.

[0157] Iqbal K, Liu F, Gong C X, Grundke-Iqbal I. Tau in Alzheimer disease and related tauopathies. Curr Alzheimer Res. 2010; 7 (8):656-64.

[0158] Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004; 25 (12):677-86.

[0159] Mushtaq G, Greig N H, Anwar F, Zamzami M A, Choudhry H, Shaik M M, Tamargo I A, Kamal M A., miRNAs as Circulating Biomarkers for Alzheimer's Disease and Parkinson's Disease. Med Chem. 2015 Oct. 30. [Epub ahead of print PMID: 26527155].

[0160] Ballard C, Gauthier S, Corbett A, Brayne C, Aarsland D, Jones E. Alzheimer's disease. Lancet 2011; 377 (9770):1019-31.

[0161] Ritter A, Cummings Fluid Biomarkers in Clinical Trials of Alzheimer's Disease Therapeutics. J. Front Neurol. 2015; 6:186.

[0162] Sandwall E, O'Callaghan P, Zhang X, Lindahl U, Lannfelt L, Li J P. Heparan sulfate mediates amyloid-beta internalization and cytotoxicity. Glycobiology 2010; 20 (5):533-41.

[0163] Snow A D, Mar H, Nochlin D, Sekiguchi R T, Kimata K, Koike Y, et al. Early accumulation of heparan sulfate in neurons and in the beta-amyloid protein-containing lesions of Alzheimer's disease and Down's syndrome. Am J Pathol. 1990; 137 (5): 1253-70.

[0164] Su J H, Cummings B J, Cotman C W. Localization of heparan sulfate glycosaminoglycan and proteoglycan core protein in aged brain and Alzheimer's disease. Neuroscience 1992; 51 (4):801-13.