| 1. |
- Ekholm Pettersson, Frida
(author)
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T-cell Differentiation and Immunological Homeostasis in Lymphopenic and Kappa Light Chain Deficient Mice
- 2002
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Doctoral thesis (other academic/artistic)abstract
- T lymphocytes are primarily involved in adaptive, cell-mediated, immune reactions. In this thesis T cells were studied regarding central and peripheral differentiation and homeostatic mechanisms for maintanance of the immune repertoire.The influence by mature T cells on thymic development was studied in C.B-17 scid/scid (SCID) mice, devoid of mature T and B cells, and whose thymocyte development is arrested at the early pro-T cell stage. When mature syngeneic T cells were injected the developmental block was overcome and there was an accumulation of CD4+CD8+ thymocytes. This event was accompanied by the maturation of medullary epithelial cells in thymus which seemed to be driven by CD8+ T cells. In the periphery there was initially a spontaneous T-cell proliferation and later, the majority of the donor T lymphocytes showed a memory phenotype with high expression of CD44 and with an early onset of proliferation and cytokine production upon stimulation. This stable pool of memory type of cells sustained for more than a year following treatment.Treating SCID mice with allogeneic T cells results in graft-versus-host disease (GVHD). Severe GVHD was dependent on the MHC-haplotype of the donor cells and was accompanied by profound alterations of the TCR-Vβ repertoire and with high production of IFN-γ.Kappa light chain (κ)-deficient mice have only half the number of B cells as their normal counterparts but normal levels of immunoglobulins. When T cells from κ-deficient mice were stimulated in vitro there was a bias towards production of B-cell stimulatory type 2 cytokines. This is proposed as a mechanism for the homeostatic control of serum immunoglobulin levels in κ-deficient mice.
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| 2. |
- Englund, Hillevi, 1980-, et al.
(author)
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Oligomerization partially explains the lowering of Aβ42 in Alzheimer's disease cerebrospinal fluid
- 2009
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In: Neuro-degenerative diseases. - : S. Karger AG. - 1660-2862 .- 1660-2854. ; 6:4, s. 139-147
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Journal article (peer-reviewed)abstract
- Background/aim: The lowering of natively analyzed Aβ42 in cerebrospinal fluid (CSF) is used as a diagnostic tool in Alzheimer’s disease (AD). Presence of Aβ oligomers can interfere with such analyses causing underestimation of Aβ levels due to epitope masking. The aim was to investigate if the lowering of CSF Aβ42 seen is caused by oligomerization. Methods: Aβ42 was analyzed under both denaturing and non-denaturing conditions. An Aβ42 oligomer ratio was calculated from these quantifications. Presence of oligomers leads to Aβ42 epitope masking during non-denaturing assays, resulting in a higher ratio. Results: The Aβ42 oligomer ratio was used for assessment of oligomerized Aβ in human CSF, after being evaluated in transgenic mouse brain homogenates. AD and mild cognitive impairment (MCI) samples displayed the expected decrease in natively measured Aβ42 compared to healthy controls and frontotemporal dementia, but not when analyzing under denaturing conditions. Accordingly, AD and MCI CSF had a higher Aβ42 oligomer ratio in CSF. Conclusion: Combining denaturing and non-denaturing quantifications of Aβ42 into an oligomer ratio enables assessment of Aβ oligomers in biological samples. The increased Aβ42 oligomer ratio for AD and MCI indicates presence of oligomers in CSF and that the lowering of natively measured Aβ42 is caused by oligomerization.
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| 3. |
- Englund, Hillevi, 1980-, et al.
(author)
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Sensitive ELISA detection of amyloid-β protofibrils in biological samples
- 2007
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In: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 103:1, s. 334-345
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Journal article (peer-reviewed)abstract
- Amyloid-β (Aβ) protofibrils are known intermediates of the in vitro Aβ aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Aβ protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Aβ protofibril immunoassay, Aβ conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Aβ protofibrils without interference from Aβ monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Aβ protofibrils in both cell and animal models, proving that Aβ protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Aβ protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Aβ protofibrils in AD and has the potential of becoming an important diagnostic assay.
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| 4. |
- Englund, Hillevi, 1980-
(author)
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Soluble amyloid-β aggregates in Alzheimer’s disease
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Soluble oligomeric aggregates of the amyloid-β (Aβ) peptide are suggested to initiate Alzheimer's disease (AD), leading to impaired synapse signalling, widespread neuronal death and loss of cognitive functions. These aggregates seem tightly linked to disease progression, and have therefore gained much attention as potential novel disease markers. In this thesis soluble oligomeric Aβ aggregates in general, and the Aβ protofibril species in particular, have been investigated with the aim to quantify and determine their role in AD pathogenesis. Sandwich-ELISAs specifically measuring Aβ42 peptides are widely used both in AD research and as complements for clinical diagnosis. Here it was demonstrated that presence of soluble Aβ aggregates disturbs such analyses, making it difficult to interpret the results. This discovery was made through analyses of samples from cell- and mouse models carrying the AD causing 'Arctic' APP mutation. When analyzed by ELISA, Aβ42 levels were reduced in Arctic samples, in contrast to levels measured by denaturing SDS-PAGE Western blot. The same divergence in Aβ42-levels between analyses was observed in CSF samples from Down syndrome infants. The discrepancy between methods was hypothesized to be due to presence of soluble Aβ aggregates leading to impaired ELISA detection caused by epitope masking. This was confirmed by developing a protofibril specific ELISA, by which samples from Arctic cell- and mouse models were demonstrated to have enhanced Aβ protofibril levels. AD patients have reduced ELISA-measured Aβ42-levels in CSF compared to healthy controls. To test if this reduction was due to oligomeric Aβ species present in AD CSF, Aβ42-levels were analyzed under both denaturing and non-denaturing conditions. These two measures were combined and an Aβ42 oligomer ratio established. Higher ratios were found in AD patients than healthy controls, implying that Aβ oligomers are present in CSF during Alzheimer pathogenesis. The observations from AD patients and young Down syndrome individuals suggest that Aβ42 oligomer formation is an early mechanism of AD pathogenesis, which potentially could be used as a biomarker to monitor disease development.
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| 5. |
- Ingelsson, Martin, et al.
(author)
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Sällsynta mutationer leder till framtidens behandling
- 2009
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In: Läkartidningen. - 0023-7205 .- 1652-7518. ; 106:20, s. 1396-1400
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Journal article (peer-reviewed)abstract
- The identification of disease-causing mutations in Alzheimer’s disease has greatly contributed to our understanding of the pathogenesis. Based on this knowledge, a number of therapeutic strategies are under development, most of which are aimed at lowering the amount of Abpeptides in the affected brain. Due to intense research efforts and massive investments at universities and in the pharmaceutical industry, the future perspectives for Alzheimer patients have never looked brighter.
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| 6. |
- Johansson, Ann-Sofi, 1978-
(author)
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Amyloid-β Protofibril Formation and Neurotoxicity : Implications for Alzheimer’s Disease
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) is the most common cause of dementia. A characteristic feature of AD is the presence of amyloid plaques in the cortex and hippocampus of the brain. The principal component of these plaques is the amyloid-β (Aβ) peptide, a cleavage product from proteolytic processing of amyloid precursor protein (APP). A central event in AD pathogenesis is the ability of Aβ monomers to aggregate into amyloid fibrils. This process involves the formation of various Aβ intermediates, including protofibrils. Protofibrils have been implicated in familial AD, as the Arctic APP mutation is associated with enhanced rate of protofibril formation in vitro. This thesis focuses on Aβ aggregation and neurotoxicity in vitro, with special emphasis on protofibril formation. Using synthetic Aβ peptides with and without the Arctic mutation, we demonstrated that the Arctic mutation accelerated both Aβ1-42 protofibril- and fibril formation, and that these processes were affected by changes in the physiochemical environment. Oxidation of Aβ methionine delayed trimer and protofibril formation in vitro. Interestingly, these oxidized peptides did not have the neurotoxic potential of their un-oxidized counterparts, suggesting that formation of trimers and further aggregation into protofibrils is necessary for the neurotoxic actions of Aβ. In agreement, stabilization of Aβ wild type protofibrils with the omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) sustained Aβ induced neurotoxicity; whereas in absence of DHA, neurotoxicity was reduced as Aβ fibrils were formed. These results suggest that the neurotoxic potential of Aβ is mainly confined to soluble aggregated forms of Aβ, not Aβ monomer/dimers or fibrillar Aβ. Stabilization of Aβ protofibrils with DHA might seem contradictory, as ω3 fatty acids generally are considered beneficial for cognition. However, we also demonstrated that DHA supplementation reduced Aβ levels in cell models of AD, providing a possible mechanism for the reported beneficial effects of DHA on cognitive measures in vivo.
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| 7. |
- Johansson, Ann-Sofi, et al.
(author)
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Docosahexaenoic acid stabilizes soluble amyloid-β protofibrils and sustains amyloid-β induced neurotoxicity in vitro
- 2007
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In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:4, s. 990-1000
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Journal article (peer-reviewed)abstract
- Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid-β (Aβ) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with Aβ, and their effect on Aβ aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble Aβ42 wild-type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid-β-induced toxicity in PC12 cells over time, whereas Aβ without docosahexaenoic acid stabilization resulted in reduced toxicity, as Aβ formed fibrils. Arachidic acid had no effect on Aβ aggregation, and neither of the fatty acids had any protofibril-stabilizing effect on Aβ42 harboring the Arctic mutation (AβE22G). Consequently, AβArctic-induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different Aβ aggregates and how endogenous lipids can affect Aβ aggregation.
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| 8. |
- Kamali-Moghaddam, Masood, et al.
(author)
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Sensitive detection of A beta protofibrils by proximity ligation : relevance for Alzheimer's disease
- 2010
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In: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 11, s. 124-
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Journal article (peer-reviewed)abstract
- Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A beta protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results: For specific detection of A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation. Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.
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| 9. |
- Lannfelt, Lars, et al.
(author)
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Translating research on brain aging into public health : a new type of immunotherapy for Alzheimer's disease
- 2010
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In: Nutrition reviews. - : Oxford University Press (OUP). - 0029-6643 .- 1753-4887. ; 68:12, s. S128-S134
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Research review (peer-reviewed)abstract
- The identification of disease-causing mutations in Alzheimer's disease has contributed greatly to the understanding of the pathogenesis of this disease. The amyloid-beta (A beta) peptide has come into focus and is believed to be central to the pathogenesis of Alzheimer's disease. With only symptomatic treatment available, efforts to develop new therapeutics aimed at lowering the amount of A beta peptides in the affected brain have intensified. In particular, immunotherapy against A beta peptides has attracted considerable interest, as it offers the possibility to generate highly specific molecules targeting highly specific moieties. Due to intense research efforts and massive investments at universities and in the pharmaceutical industry, the outlook for patients and their relatives has never been brighter.
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| 10. |
- Lord, Anna, 1979-, et al.
(author)
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Amyloid-β protofibril levels correlate with spatial learning in Arctic Alzheimer’s disease transgenic mice
- 2009
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In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:4, s. 995-1006
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Journal article (peer-reviewed)abstract
- Oligomeric assemblies of amyloid-β (Aβ) are suggested to be central in the pathogenesis of Alzheimer's disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long-term potentiation. The tg-ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg-ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg-ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg-ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg-ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg-ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age-dependent manner in tg-ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.
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