| 1001. |
- Mersmann, Michael, et al.
(författare)
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Towards proteome scale antibody selections using phage display
- 2010
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Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 27:2, s. 118-128
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Tidskriftsartikel (refereegranskat)abstract
- In vitro antibody generation by panning a large universal gene library with phage display was employed to generate antibodies to more than 60 different antigens. Of particular interest was a comparison of pannings on 20 different SH2 domains provided by the Structural Genomics Consortium (SGC). Streamlined methods for high throughput antibody generation developed within the 'Antibody Factory' of the German National Genome Research Network (NGFN) were demonstrated to minimise effort and provide a reliable and robust source for antibodies. For the SH2 domains, in two successive series of selections, 2668 clones were analysed, resulting in 347 primary hits in ELISA. Half of these hits were further analysed, and more than 90 different scFv antibodies to all antigens were identified. The validation of selected antibodies by cross-reactivity ELISA, western blot and on protein microarrays demonstrated the versatility of the in vitro antibody selection pipeline to generate a renewable resource of highly specific monoclonal binders in proteome scale numbers with substantially reduced effort and time.
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| 1002. |
- Mescia, Federica, et al.
(författare)
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Autoantibody Profiling and Anti-Kinesin Reactivity in ANCA-Associated Vasculitis
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:20
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Tidskriftsartikel (refereegranskat)abstract
- ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.
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| 1003. |
- Michno, Wojciech, 1992, et al.
(författare)
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Multimodal Chemical Imaging of Amyloid Plaque Polymorphism Reveals A beta Aggregation Dependent Anionic Lipid Accumulations and Metabolism
- 2018
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:13, s. 8130-8138
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid plaque formation constitutes one of the main pathological hallmarks of Alzheimer's disease (AD) and is suggested to be a critical factor driving disease pathogenesis. Interestingly, in patients that display amyloid pathology but remain cognitively normal, A beta deposits are predominantly of diffuse morphology suggesting that cored plaque formation is primarily associated with cognitive deterioration and AD pathogenesis. Little is known about the molecular mechanism responsible for conversion of monomeric A beta into neurotoxic aggregates and the predominantly cored deposits observed in AD. The structural diversity among A beta plaques, including cored/compact- and diffuse, may be linked to their distinct A beta profile and other chemical species including neuronal lipids. We developed a novel, chemical imaging paradigm combining matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and fluorescent amyloid staining. This multimodal imaging approach was used to probe the lipid chemistry associated with structural plaque heterogeneity in transgenic AD mice (tgAPP(Swe)) and was correlated to A beta profiles determined by subsequent laser microdissection and immunoprecipitation-mass spectrometry. Multivariate image analysis revealed an inverse localization of ceramides and their matching metabolites to diffuse and cored structures within single plaques, respectively. Moreover, phosphatidylinositols implicated in AD pathogenesis, were found to localize to the diffuse A beta structures and correlate with A beta 1-42. Further, lysophospholipids implicated in neuroinflammation were increased in all A beta deposits. The results support previous clinical findings on the importance of lipid disturbances in AD pathophysiology and associated sphingolipid processing. These data highlight the potential of multimodal imaging as a powerful technology to probe neuropathological mechanisms.
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| 1004. |
- Michno, Wojciech, 1992, et al.
(författare)
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Pyroglutamation of amyloid-βx-42 (Aβx-42) followed by Aβ1–40 deposition underlies plaque polymorphism in progressing Alzheimer's disease pathology
- 2019
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Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 294:17, s. 6719-6732
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid- (A) pathology in Alzheimer's disease (AD) is characterized by the formation of polymorphic deposits comprising diffuse and cored plaques. Because diffuse plaques are predominantly observed in cognitively unaffected, amyloid-positive (CU-AP) individuals, pathogenic conversion into cored plaques appears to be critical to AD pathogenesis. Herein, we identified the distinct A species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated A polymorphism with amyloid conformation-sensitive dyes and a novel in situ MS paradigm for chemical characterization of hyperspectrally delineated plaque morphotypes. We found that maturation of diffuse into cored plaques correlated with increased A1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that A1-40 aggregates at the core structure of mature plaques, whereas A1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Ax-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating A1-40 deposition. Experiments in tgAPP(Swe) mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of A1-42 and that A plaque maturation over time is associated with increases in A1-40. Finally, we found that A1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Ax-42 pyroglutamation and A1-40 deposition are critical events in priming and maturation of pathogenic A from diffuse into cored plaques, underlying neurotoxic plaque development in AD.
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| 1005. |
- Midlöv, Patrik, et al.
(författare)
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PERson-centredness in hypertension management using information technology (PERHIT): a protocol for a randomised controlled trial in primary health care
- 2020
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Ingår i: Blood Pressure. - : TAYLOR & FRANCIS LTD. - 0803-7051 .- 1651-1999. ; 29:3, s. 149-156
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: For primary health care (PHC), hypertension is the number one diagnosis for planned health care visits. The treatment of high blood pressure (BP) and its consequences constitutes a substantial economic burden. In spite of efficient antihypertensive medications, a low percentage of patients reach a well-controlled BP. The PERson-centredness in Hypertension management using Information Technology (PERHIT) Study is a multicentre randomised controlled trial. PERHIT is designed to evaluate the effect of supporting self-management on systolic blood pressure by the use of information technology in Swedish primary health care. Materials and Methods: After inclusion, 900 patients from 36 PHC centres are randomised to two groups. In the intervention group, patients are provided with a self-management support system including a home-BP monitor and further requested to perform self-reports and measure BP every evening for eight consecutive weeks. In the control group, patients receive treatment as usual. Results: The primary outcome will be the change in systolic blood pressure in patients with hypertension. In addition, person-centredness, daily life activities, awareness of risk and health care costs will also be evaluated. Conclusion: The results of this randomised controlled trial with assessment of blood pressure and same-day self-reports will provide patients a tool to understand the interplay between blood pressure and lifestyle applicable to primary health care. The self-management support system may be of importance for improved adherence to treatment and persistence to treatment recommendations.
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| 1006. |
- Mikus, Maria, et al.
(författare)
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Allergome-wide peptide microarrays enable epitope deconvolution in allergen-specific immunotherapy
- 2020
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Ingår i: Journal of Allergy and Clinical Immunology. - : Mosby Inc.. - 0091-6749 .- 1097-6825.
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Tidskriftsartikel (refereegranskat)abstract
- Background: The interaction of allergens and allergen-specific IgE initiates the allergic cascade after crosslinking of receptors on effector cells. Antibodies of other isotypes may modulate such a reaction. Receptor crosslinking requires binding of antibodies to multiple epitopes on the allergen. Limited information is available on the complexity of the epitope structure of most allergens. Objectives: We sought to allow description of the complexity of IgE, IgG4, and IgG epitope recognition at a global, allergome-wide level during allergen-specific immunotherapy (AIT). Methods: We generated an allergome-wide microarray comprising 731 allergens in the form of more than 172,000 overlapping 16-mer peptides. Allergen recognition by IgE, IgG4, and IgG was examined in serum samples collected from subjects undergoing AIT against pollen allergy. Results: Extensive induction of linear peptide-specific Phl p 1– and Bet v 1–specific humoral immunity was demonstrated in subjects undergoing a 3-year-long AIT against grass and birch pollen allergy, respectively. Epitope profiles differed between subjects but were largely established already after 1 year of AIT, suggesting that dominant allergen-specific antibody clones remained as important contributors to humoral immunity following their initial establishment during the early phase of AIT. Complex, subject-specific patterns of allergen isoform and group cross-reactivities in the repertoires were observed, patterns that may indicate different levels of protection against different allergen sources. Conclusions: The study highlights the complexity and subject-specific nature of allergen epitopes recognized following AIT. We envisage that epitope deconvolution will be an important aspect of future efforts to describe and analyze the outcomes of AIT in a personalized manner.
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| 1007. |
- Mikus, Maria, 1986-
(författare)
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Array-based identification of disease-associated proteins
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To increase our understanding of the human body in both health and disease, proteins can be studied in samples such as plasma and serum to provide a molecular profile of the physiological status. In the work presented in this thesis, array-based methods were used to study associations of protein and autoantibody profiles with disease. The methods included antibody suspension bead arrays for protein profiling and planar antigen arrays or antigen suspension bead arrays for autoantibody profiling.In Paper I, we studied protein levels in the context of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We identified three proteins, NEFM, RGS18 and SLC25A20, to be significantly elevated in patients with ALS. We also evaluated the diagnostic potential of these proteins, reaching areas under the curves (AUCs) between 0.78 and 0.86 for each of the three proteins individually.In Paper II, drug-induced liver injury (DILI) cases and controls were studied in four independent cohorts of longitudinal and cross-sectional design and covering a range of drugs. The protein FABP1 was elevated in DILI cases upon initiation of treatment whereas CDH5 were elevated before treatment. Furthermore, we compared FABP1 with the clinically measured alanine aminotransferase (ALT), and identified some aspects in which FABP1 was superior: tissue distribution – FABP1 was not found in skeletal and heart muscle tissue, injuries in which can cause elevations of ALT; kinetics – FABP1 is smaller and has a lower half-life compared to ALT. Both of these circumstances mean that FABP1 as a biomarker has the potential to more accurately reflect ongoing injury.In Paper III, asthma of different severities, chronic obstructive pulmonary disease and healthy controls from two independent cohorts were studied. The levels of ten proteins were verified to be significantly elevated in severe asthma compared to both mild-to-moderate asthma and healthy controls in both cohorts. We also clustered asthma patients based on their protein profiles and identified six subgroups that could help to guide the appropriate treatment.In Paper IV, atopic dermatitis (AD) of different severities and healthy controls were studied. Increased autoantibody reactivity to four antigens, KRTAP17-1, HSPA4, S100A12 and S100Z, were observed in AD patients or in any of the two severity disease subgroups compared to controls.In summary, the work included in this thesis highlights the applicability of protein array-based methods in various contexts and in studying various research questions. Disease-associated proteins were identified and further studies will determine their utility.
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| 1008. |
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| 1009. |
- Mikus, Maria, et al.
(författare)
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Elevated levels of circulating CDH5 and FABP1 in association with human drug-induced liver injury
- 2016
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Ingår i: Liver international. - : John Wiley & Sons. - 1478-3223 .- 1478-3231. ; 37:1, s. 132-140
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Tidskriftsartikel (refereegranskat)abstract
- Background & Aims: The occurrence of drug-induced liver injury (DILI) is a major issue in all phases of drug development. To identify novel biomarker candidates associated with DILI, we utilised an affinity proteomics strategy, where antibody suspension bead arrays were applied to profile plasma and serum samples from human DILI cases and controls. Methods: An initial screening was performed using 4594 randomly selected antibodies, representing 3450 human proteins. Resulting candidate proteins together with proposed DILI biomarker candidates generated a DILI array of 251 proteins for subsequent target analysis and verifications. In total, 1196 samples from 241 individuals across four independent cohorts were profiled: healthy volunteers receiving acetaminophen, patients with human immunodeficiency virus and/or tuberculosis receiving treatment, DILI cases originating from a wide spectrum of drugs, and healthy volunteers receiving heparins. Results: We observed elevated levels of cadherin 5, type 2 (CDH5) and fatty acid-binding protein 1 (FABP1) in DILI cases. In the two longitudinal cohorts, CDH5 was elevated already at baseline. FABP1 was elevated after treatment initiation and seemed to respond more rapidly than alanine aminotransferase (ALT). The elevations were verified in the DILI cases treated with various drugs. In the heparin cohort, CDH5 was stable over time whereas FABP1 was elevated. Conclusions: These results suggest that CDH5 may have value as a susceptibility marker for DILI. FABP1 was identified as a biomarker candidate with superior characteristics regarding tissue distribution and kinetics compared to ALT but likely with limited predictive value for the development of severe DILI. Further studies are needed to determine the clinical utility of the proposed markers.
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| 1010. |
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