Киберпространство и виртуальная реальность: попытка историко-философского анализа

Материалы IV Республик. науч. конф. студентов, магистрантов и аспирантов, Гомель, 12 мая 2011 г

Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ 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 Aβ 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.</p> <p>Results</p> <p>For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ 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 Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ 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 Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.</p> <p>Conclusions</p> <p>The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.</p

T-cell Differentiation and Immunological Homeostasis in Lymphopenic and Kappa Light Chain Deficient Mice

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

Large aggregates are the major soluble Aβ species in AD brain fractionated with density gradient ultracentrifugation.

Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80-500 kDa synthetic Aβ protofibrils and were equally detected with mAb158

Increased Number of Plasma B Cells Producing Autoantibodies Against A beta(42) Protofibrils in Alzheimer's Disease

The Alzheimer's disease (AD)-related peptide amyloid-beta (A beta) has a propensity to aggregate into various assemblies including toxic soluble A beta protofibrils. Several studies have reported the existence of anti-A beta antibodies in humans. However, it is still debated whether levels of anti-A beta antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma A beta makes it difficult to reliably measure the concentration of circulating anti-A beta antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-A beta antibody production on a cellular level by measuring the amount of anti-A beta antibody producing cells instead of the plasma level of anti-A beta antibodies. To our knowledge, this is the first time the anti-A beta antibody response in plasma has been compared in AD patients and age-matched healthy individuals using the enzyme-linked immunospot (ELISpot) technique. Both AD patients and healthy individuals had low levels of B cells producing antibodies binding A beta(40) monomers, whereas the number of cells producing antibodies toward A beta(42) protofibrils was higher overall and significantly higher in AD compared to healthy controls. This study shows, by an alternative and reliable method, that there is a specific immune response to the toxic A beta protofibrils, which is significantly increased in AD patients

Fractionation of transgenic mouse brain extracts.

<p>Fractions of mouse brain homogenate from AβPP_ArcSwe transgenic mice (n = 5) and non-transgenic littermates (n = 5) were analyzed with Aβ1–42 (A), Aβ1–40 (B) and Aβ protofibril specific (C) ELISAs. Horizontal lines indicate the mean value of each group. Immunoprecipitation of pooled material from fraction 2 of transgenic mouse brain homogenate with the conformation specific Aβ protofibril selective antibody mAb158 covalently coupled to Dynabeads (0 – non immunoprecipitated sample, IP – immunoprecipitated material, S – supernatant remaining after ip) (D). Error bars indicate the standard deviation.</p

Fractionation of human brain extracts.

<p>Fractions of human brain homogenate from temporal cortex of diseased AD patients (n = 7, including one AβPP_Swe and one AβPP_Arc mutation carrier) and non-AD subjects (n = 4, one control subject and three FTD patients) were analyzed with Aβx-42 (A), Aβx-40 (B), Aβ1–42 (C) and Aβ1–40 (D) ELISAs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032014#s2" target="_blank">Results</a> from the individual carrying the Swedish mutation is marked ‘Swe’ in the Aβx-40 and Aβ1–40 graphs. Horizontal lines indicate the mean value of each group. The level of N-terminal truncation of Aβ42 was determined as a ratio between Aβ1–42 and Aβx-42 (1-[Aβ1–42]/[Aβx-42]) (E), with error bars indicating the standard deviation. Immunoprecipitation of pooled material from fraction 2 of AD brain and non-AD brain with the conformation specific Aβ protofibril selective antibody mAb158 covalently coupled to Dynabeads (0 – non immonuprecipitated sample, IP – immunoprecipitated material, S – supernatant remaining after ip) (F). Error bars indicate the standard deviation.</p

Summary of cases.

<p>Summary of cases.</p