| 1. |
- Gobom, Johan, et al.
(författare)
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Antibody-free measurement of cerebrospinal fluid tau phosphorylation across the Alzheimer's disease continuum.
- 2022
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Ingår i: Molecular neurodegeneration. - : Springer Science and Business Media LLC. - 1750-1326. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease is characterized by an abnormal increase of phosphorylated tau (pTau) species in the CSF. It has been suggested that emergence of different pTau forms may parallel disease progression. Therefore, targeting multiple specific pTau forms may allow for a deeper understanding of disease evolution and underlying pathophysiology. Current immunoassays measure pTau epitopes separately and may capture phosphorylated tau fragments of different length depending on the non-pTau antibody used in the assay sandwich pair, which bias the measurement.We developed the first antibody-free mass spectrometric method to simultaneously measure multiple phosphorylated epitopes in CSF tau: pT181, pS199, pS202, pT205, pT217, pT231, and pS396. The method was first evaluated in biochemically defined Alzheimer's disease and control CSF samples (n=38). All seven pTau epitopes clearly separated Alzheimer's disease from non-AD (p<0.001, AUC=0.84-0.98). We proceeded with clinical validation of the method in the TRIAD (n=165) and BioFINDER-2 cohorts (n=563), consisting of patients across the full Alzheimer's disease continuum, including also young controls (<40years), as well as patients with frontotemporal dementia and other neurodegenerative disorders.Increased levels of all phosphorylated epitopes were found in Alzheimer's disease dementia and Aβ positron emission tomography-positive patients with mild cognitive impairment compared with Aβ-negative controls. For Alzheimer's disease dementia compared with Aβ-negative controls, the best biomarker performance was observed for pT231 (TRIAD: AUC=98.73%, fold change=7.64; BioFINDER-2: AUC=91.89%, fold change=10.65), pT217 (TRIAD: AUC=99.71%, fold change=6.33; BioFINDER-2: AUC=98.12%, fold change=8.83) and pT205 (TRIAD: AUC=99.07%, fold change=5.34; BioFINDER-2: AUC=93.51%, fold change=3.92). These phospho-epitopes also discriminated between Aβ-positive and Aβ-negative cognitively unimpaired individuals: pT217 (TRIAD: AUC=83.26, fold change=2.39; BioFINDER-2: AUC=91.05%, fold change=3.29), pT231 (TRIAD: AUC=86.25, fold change=3.80; BioFINDER-2: AUC=78.69%, fold change=3.65) and pT205 (TRIAD: AUC=71.58, fold change=1.51; BioFINDER-2: AUC=71.11%, fold change=1.70).While an increase was found for all pTau species examined, the highest fold change in Alzheimer's disease was found for pT231, pT217 and pT205. Simultaneous antibody-free measurement of pTau epitopes by mass spectrometry avoids possible bias caused by differences in antibody affinity for modified or processed forms of tau, provides insights into tau pathophysiology and may facilitate clinical trials on tau-based drug candidates.
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| 2. |
- Karikari, Thomas, et al.
(författare)
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Blood phospho-tau in Alzheimer disease: analysis, interpretation, and clinical utility
- 2022
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Ingår i: Nature Reviews Neurology. - : Springer Science and Business Media LLC. - 1759-4758 .- 1759-4766. ; 18, s. 400-418
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Tidskriftsartikel (refereegranskat)abstract
- Recent technological advances have enabled the detection of specific forms of phosphorylated tau in blood. Here, the authors summarize the performance of blood phosphorylated tau biomarkers in the context of Alzheimer disease and highlight related ethical, analytical and clinical challenges. Well-authenticated biomarkers can provide critical insights into the biological basis of Alzheimer disease (AD) to enable timely and accurate diagnosis, estimate future burden and support therapeutic trials. Current cerebrospinal fluid and molecular neuroimaging biomarkers fulfil these criteria but lack the scalability and simplicity necessary for widespread application. Blood biomarkers of adequate effectiveness have the potential to act as first-line diagnostic and prognostic tools, and offer the possibility of extensive population screening and use that is not limited to specialized centres. Accelerated progress in our understanding of the biochemistry of brain-derived tau protein and advances in ultrasensitive technologies have enabled the development of AD-specific phosphorylated tau (p-tau) biomarkers in blood. In this Review we discuss how new information on the molecular processing of brain p-tau and secretion of specific fragments into biofluids is informing blood biomarker development, enabling the evaluation of preanalytical factors that affect quantification, and informing harmonized protocols for blood handling. We also review the performance of blood p-tau biomarkers in the context of AD and discuss their potential contexts of use for clinical and research purposes. Finally, we highlight outstanding ethical, clinical and analytical challenges, and outline the steps that need to be taken to standardize inter-laboratory and inter-assay measurements.
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| 3. |
- Mila-Aloma, M., et al.
(författare)
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Plasma p-tau231 and p-tau217 as state markers of amyloid-beta pathology in preclinical Alzheimer's disease
- 2022
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 28, s. 1797-1801
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Tidskriftsartikel (refereegranskat)abstract
- A comprehensive comparison of Alzheimer's disease blood biomarkers in cognitively unimpaired individuals reveals that plasma p-tau231 and p-tau217 capture very early A beta changes, showing promise as markers to enrich a preclinical population for Alzheimer's disease clinical trials Blood biomarkers indicating elevated amyloid-beta (A beta) pathology in preclinical Alzheimer's disease are needed to facilitate the initial screening process of participants in disease-modifying trials. Previous biofluid data suggest that phosphorylated tau231 (p-tau231) could indicate incipient A beta pathology, but a comprehensive comparison with other putative blood biomarkers is lacking. In the ALFA+ cohort, all tested plasma biomarkers (p-tau181, p-tau217, p-tau231, GFAP, NfL and A beta 42/40) were significantly changed in preclinical Alzheimer's disease. However, plasma p-tau231 reached abnormal levels with the lowest A beta burden. Plasma p-tau231 and p-tau217 had the strongest association with A beta positron emission tomography (PET) retention in early accumulating regions and associated with longitudinal increases in A beta PET uptake in individuals without overt A beta pathology at baseline. In summary, plasma p-tau231 and p-tau217 better capture the earliest cerebral A beta changes, before overt A beta plaque pathology is present, and are promising blood biomarkers to enrich a preclinical population for Alzheimer's disease clinical trials.
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| 4. |
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| 5. |
- Montoliu-Gaya, Laia, et al.
(författare)
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Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris
- 2022
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Ingår i: Therapeutic Antibodies. Methods and Protocols. Gunnar Houen (red.). - New York, NY : Springer. - 1064-3745. - 9781071614495 ; , s. 151-167
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The interest in the use of monoclonal antibodies as therapeutic molecules has raised in the recent years. Due to their high affinity and specificity towards other biological molecules, antibodies are being widely used to treat a broad range of human diseases such as cancer, rheumatism, and cardiovascular diseases. Currently, the production of IgG-like antibodies is mainly obtained from stable or transient mammalian expression systems that allow proper folding and posttranslational modifications. Despite the technological advances of the last decade, the use of these systems still has a rather high production cost and long processing times. For these reasons, researchers are increasingly interested in alternative antibody production methods as well as alternative antibody formats. Bacterial systems, such as Escherichia coli, are extensively being used for recombinant protein production because their easy manipulation and cheap costs. However, the presence of lipopolysaccharides (LPS) traces in the already fractionated recombinant protein makes these systems not good candidates for the preparation of therapeutic molecules. Yeast systems, such as Pichia pastoris, present the convenient easy manipulation of microbial systems but show some key advantages of eukaryotic expression systems, like improved folding machinery and absence of LPS. They are especially suitable for the production of antibody fragments, which do not need human-like glycosylation, avoiding the high costs of mammalian systems. Here, the protocol for the expression and purification of a single-chain antibody fragment (scFv) in P. pastoris is provided, in deep detail for lab manipulation and briefly for a 5L-bioreactor production. © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
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| 6. |
- Nilsson, Johanna, 1993, et al.
(författare)
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Quantification of SNAP-25 with mass spectrometry and Simoa: a method comparison in Alzheimer's disease
- 2022
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Ingår i: Alzheimer's Research & Therapy. - : Springer Science and Business Media LLC. - 1758-9193. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Synaptic dysfunction and degeneration are central to Alzheimer's disease (AD) and have been found to correlate strongly with cognitive decline. Thus, studying cerebrospinal fluid (CSF) biomarkers reflecting synaptic degeneration, such as the presynaptic protein synaptosomal-associated protein 25 (SNAP-25), is of importance to better understand the AD pathophysiology. Methods We compared a newly developed Single molecule array (Simoa) immunoassay for SNAP-25 with an in-house immunoprecipitation mass spectrometry (IP-MS) method in a well-characterized clinical cohort (n = 70) consisting of cognitively unimpaired (CU) and cognitively impaired (CI) individuals with and without A beta pathology (A beta+ and A beta-). Results A strong correlation (Spearman's rank correlation coefficient (r(s)) > 0.88; p < 0.0001) was found between the Simoa and IP-MS methods, and no statistically significant difference was found for their clinical performance to identify AD pathophysiology in the form of A beta pathology. Increased CSF SNAP-25 levels in CI A beta+ compared with CU A beta- (Simoa, p <= 0.01; IP-MS, p <= 0.05) and CI A beta- (Simoa, p <= 0.01; IP-MS, p <= 0.05) were observed. In independent blood samples (n = 32), the Simoa SNAP-25 assay was found to lack analytical sensitivity for quantification of SNAP-25 in plasma. Conclusions These results indicate that the Simoa SNAP-25 method can be used interchangeably with the IP-MS method for the quantification of SNAP-25 in CSF. Additionally, these results confirm that CSF SNAP-25 is increased in relation to amyloid pathology in the AD continuum.
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| 7. |
- Roda, A. R., et al.
(författare)
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Amyloid-beta peptide and tau protein crosstalk in Alzheimer's disease
- 2022
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Ingår i: Neural Regeneration Research. - : Medknow. - 1673-5374. ; 17:8, s. 1666-1674
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease is a neurodegenerative disease that accounts for most of the 50-million dementia cases worldwide in 2018. A large amount of evidence supports the amyloid cascade hypothesis, which states that amyloid-beta accumulation triggers tau hyperphosphorylation and aggregation in form of neurofibrillary tangles, and these aggregates lead to inflammation, synaptic impairment, neuronal loss, and thus to cognitive decline and behavioral abnormalities. The poor correlation found between cognitive decline and amyloid plaques, have led the scientific community to question whether amyloid-beta accumulation is actually triggering neurodegeneration in Alzheimer's disease. The occurrence of tau neurofibrillary tangles better correlates to neuronal loss and clinical symptoms and, although amyloid-beta may initiate the cascade of events, tau impairment is likely the effector molecule of neurodegeneration. Recently, it has been shown that amyloid-beta and tau cooperatively work to impair transcription of genes involved in synaptic function and, more importantly, that downregulation of tau partially reverses transcriptional perturbations. Despite mounting evidence points to an interplay between amyloid-beta and tau, some factors could independently affect both pathologies. Thus, the dual pathway hypothesis, which states that there are common upstream triggers causing both amyloid-beta and tau abnormalities has been proposed. Among others, the immune system seems to be strongly involved in amyloid-beta and tau pathologies. Other factors, as the apolipoprotein E epsilon 4 isoform has been suggested to act as a link between amyloid-beta and tau hyperphosphorylation. Interestingly, amyloid-beta-immunotherapy reduces not only amyloid-beta but also tau levels in animal models and in clinical trials. Likewise, it has been shown that tau-immunotherapy also reduces amyloid-beta levels. Thus, even though amyloid-beta immunotherapy is more advanced than tau-immunotherapy, combined amyloid-beta and tau-directed therapies at early stages of the disease have recently been proposed as a strategy to stop the progression of Alzheimer's disease.
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