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Selection of transthyretin amyloid inhibitorsIakovleva, Irina January 2016 (has links)
Amyloidosis is a group of clinical disorders caused by the aggregation of specific proteins into abnormal extracellular deposits. Today, 31 different proteins have been linked to amyloid diseases including transthyretin-related amyloidosis (ATTR). ATTR occurs through the aggregation of either wild-type plasma protein transthyretin (TTR) or a mutated form. TTR is a homotetramer that under normal circumstances functions as a carrier of thyroxine and retinol binding protein. The aggregation cascade requires dissociation of the tetramer into monomers, and preventing this dissociation represents a potential mode of intervention. Interestingly, small molecules, referred as kinetic stabilizers, can bind to TTR’s thyroxine-binding site (TBS) and such molecules are currently being used as a therapeutic approach to impair tetramer dissociation. The efficacy of TTR stabilization is directly correlated to the binding affinity of the ligand to TBS. However, the binding of the ligand to TTR in vivo can be affected by other plasma components resulting in poor efficacy. Thus, the selectivity of ligands is an important parameter. We have designed an assay where the ability to stabilize TTR can be directly evaluated in plasma and we have investigated the stabilizing effect of nine potential TTR binders (Paper I). The results, surprisingly, revealed that the binding affinity of molecules has a poor correlation to its selectivity. However, the nature of protein-ligand complex formation can also be described by enthalpic (∆H) and entropic (∆S) energy contributions. ∆H represents the change in chemical bonds and frequently requires a higher order of orientation compared to the ∆S component, which mainly represents the hydrophobic effect via the exclusion of water. We hypothesized that ligands possessing high ΔH in binding to their co-partner would also be more specific in a complex environment such as plasma. By applying a thermodynamic analysis using isothermal titration calorimetry, we found that the selectivity in plasma correlates well with the ∆H contribution and might, therefore, be a better predictor for selectivity. Luteolin was found to be a highly selective stabilizer of TTR and was investigated further (Paper II). The ligand displayed a significant rescuing effect in both cell culture and animal models. However, luteolin undergoes rapid enzymatic degradation in the liver and this impairs its use as a potential therapeutic drug. To attempt to circumvent this issue, we modified the most exposed hydroxyl group thus rendering the molecule inert towards glucuronidation (Paper III). The substitutions resulted in higher stability in the face of hepatic degradation molecules, but they also affected the selectivity in a negative manner. The screening for new TTR stabilizers resulted in the discovery of tetrabromobisphenol A, which displayed a very high selectivity (Paper IV). This study also included a comparison with the drug Vyndaqel™ which currently is in clinically use, and showed how the dosage could be altered to acquire a better level of saturation and possibly also a better clinical effect. Taken together we present new molecules with the ability to stabilize TTR, and these can serve as scaffolds for the design of new drugs. We present a method to measure the efficacy of a TTR-stabilizing drugs in a complex matrix and as well as a way to adjust the dosage of existing drugs. We also show that the selectivity of a drug is affected by the relative proportion of ∆H and ∆S, and this is of interest for drug design in general.
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Cardiac function in hereditary transthyretin amyloidosis : an echocardiographic study / Hjärtfunktion vid ärftlig transtyretin-amyloidos : en ekokardiografisk studieArvidsson, Sandra January 2016 (has links)
Background: Hereditary transthyretin amyloidosis (ATTR) is a lethal disease in which misfolded transthyretin (TTR) proteins accumulate as insoluble aggregates in tissues throughout the body. A common mutation is the exchange of valine to methionine at place 30 (TTR V30M), a form endemically found in the northern parts of Sweden. The main treatment option for ATTR amyloidosis is liver transplantation as the procedure halts production of mutated transthyretin. The disease is associated with marked phenotypic diversity ranging from predominant cardiac complications to pure neuropathy. Two different types of fibril composition – one in which both fragmented and full-length TTR are present (type A) and one consisting of only full-length TTR (type B) have been suggested to account for some phenotypic differences. Cardiac amyloidosis is associated with increased myocardial thickness and the disease could easily be mistaken for other entities characterised by myocardial thickening, such as sarcomeric hypertrophic cardiomyopathy (HCM). The aims in this thesis were to investigate echocardiographic characteristics in Swedish ATTR amyloidosis patients, and to identify markers aiding in differentiating ATTR heart disease from HCM. Another objective was to examine the impact of fibril composition and sex on the phenotypic variation in amyloid heart disease. Methods: A total of 122 ATTR amyloidosis patients that had undergone thorough echocardiographic examinations were included in the studies. Analyses of ventricular geometry as well as assessment of systolic and diastolic function were performed, using both conventional echocardiographic methods and speckle tracking technique. ECG analysis was conducted in study I, allowing measurement of QRS voltage. In study I and study II ATTR patients were compared to patients with HCM. In addition, 30 healthy controls were added to study II. Results: When parameters from ECG and echocardiography were investigated, the results revealed that the combination of QRS voltage <30 mm (<3 mV) and an interventricular/posterior wall thickness quotient <1.6 could differentiate cardiac ATTR amyloidosis from HCM. Differences in degree of right ventricular involvement were also demonstrated between HCM and ATTR amyloidosis, where ATTR patients displayed a right ventricular apical sparing pattern whereas the inverse pattern was found in HCM. Analysis of fibril composition revealed increased LV wall thickness in type A patients compared to type B, but in addition type A women displayed both lower myocardial thickness and more preserved systolic function as compared to type A males. When cardiac geometry and function were evaluated pre and post liver transplantation in type A and B patients, significant deterioration was detected in type A but not in type B patients after liver transplantation. Conclusions: Increasing awareness of typical cardiac amyloidotic signs by echocardiography is important to reduce the risk of delayed diagnosis. Our classification model based on ECG and echocardiography could aid in differentiating ATTR amyloidosis from HCM. Furthermore, the apical sparing pattern found in the right ventricle may pose another clue for amyloid heart disease, although it requires to be studied further. Furthermore, we disclosed that type A fibrils, male sex and increasing age were important determinants of increased myocardial thickness. As type A fibril patients displayed rapid cardiac deterioration after liver transplantation other treatment options should probably be sought for this group of patients.
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Preparation of monoclonal antibodies against immunoglobulin kappa of AL-amyloidosis and characterization of antibody producing hybridoma cellsHossain, Ishrat January 2017 (has links)
No description available.
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Characterization of monoclonal antibodies reactivity patterns against transthyretinEdberg, Kristina January 2024 (has links)
Amyloidosis is a disease caused by misfolding of proteins that accumulate in different organs in the body. One of the most common forms of amyloidosis is Transthyretin amyloidosis (ATTR-amyloidosis). Diagnostic of amyloidosis is done by Congo red staining and immunohistochemistry where high-affinity monoclonal antibodies are preferred. For treatment to be effective it is necessary to obtain the diagnosis at an early stage. The aim of this study was to produce monoclonal antibodies against transthyretin for diagnostic purposes of ATTR-amyloidosis. To produce monoclonal antibodies, the spleen from a mouse immunized with transthyretin was collected. Antibody producing cells from the spleen were fused with myeloma cells deficient of their own antibody production. After 2 weeks culture in HAT selection media cells were screened for antibody production with ELISA and immune-histochemistry. Recombinant transtyretin (TTR) was produced and used as antigen in the ELISA. All 65 hybridomas tested were negative on ELISA. Three out of 25 hybridomas tested in immunohistochemistry showed visible staining against muscle cells in tissue from patients with TTR amyloid deposits. No antibody could be produced that detected ATTR-amyloidosis deposits.
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Defining cellular and molecular mechanisms of hereditary transthyretin amyloidosisGiadone, Richard Michael 29 May 2020 (has links)
Hereditary transthyretin amyloidosis (ATTR amyloidosis) is a multi-system protein folding disorder that results from >100 described mutations in the transthyretin (TTR) gene. In the disease, non-natively folded TTR, originally produced by the liver, travels throughout circulation and deposits extracellularly at downstream target organs. The multi-tissue etiology of the disease makes it difficult to study in vitro, while no mouse model accurately recapitulates disease pathology. Therefore, we utilized patient-specific induced pluripotent stem cells (iPSCs) to test the hypothesis that production of and exposure to destabilized TTRs results in distinct cellular and molecular changes.
The liver’s contribution to the deposition of TTR at distal tissues is understudied. As a result, in Aim 1 we sought to assess the effects of destabilized TTR production on effector hepatic cells. To this end, we utilized gene editing to generate isogenic, patient iPSCs expressing either mutant or wild-type TTR. Combining this tool with single cell RNAseq, we identified hepatic proteostasis factors, including unfolded protein response (UPR) pathways, whose expression coincided with the production of destabilized TTR. Enhancing endoplasmic reticulum (ER) proteostasis within patient hepatic cells via exogenous activation of adaptive UPR signaling, we demonstrated preferential reduction in the secretion of pathogenic TTR. In turn, we demonstrated that production of disease-associated TTR correlates with expression of proteostasis factors capable of regulating TTR secretion and in turn downstream pathogenesis.
ATTR amyloidosis patients exhibit extreme phenotypic variation (e.g. TTR fibril deposits at cardiac tissue and/or peripheral nerves). In Aim 2, we sought to define responses of target cell types to pathologically-diverse TTRs. To accomplish this, we profiled transcriptomic changes resulting from exposure to a variety of destabilized TTRs to determine 1) target cell response to TTR exposure and 2) how this response changes across diverse variants and cell types. In doing so, we found that TTR exposure elicits distinct variant- and cell type-specific transcriptional responses.
Herein, we addressed our central hypothesis by profiling destabilized TTR production within hepatic cells and TTR exposure at target cell types. Collectively, these data may result in the discovery of unidentified and potentially druggable pathologically-associated pathways for ATTR amyloidosis and other systemic amyloid diseases.
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Feasibility of quantitative 99mTc-DPD scintigraphy SPECT/CT for assessing the burden of ATTR cardiac amyloidosis / Genomförbarhet av kvantitativ 99mTc-DPD-scintigrafi med SPECT/CT för att bedöma svårighetsgraden av ATTR hjärtamyloidosKhalaf, Sajad Kadhim January 2023 (has links)
Background: Amyloid transthyretin (ATTR) cardiomyopathy is caused by the deposition of misfolded proteins, known as amyloid fibrils, in the myocardium. Quantitative Single Photon Emission Computed Tomography (QSPECT) utilizing 99mTc-DPD scintigraphy has the potential to assess ATTR-suspected cardiac amyloidosis (CA). This method could offer improved risk stratification and therapy response monitoring compared to the established Perugini score system. The primary objective of this study is to evaluate the feasibility of employing a quantitative approach and correlating various parameters with LVMI (left ventricular mass index). Method: Initially, planar and volumetric sensitivity measurements were conducted, followed by verification of accuracy measurements. Several torso phantom acquisitions were then performed to evaluate the accuracy and repeatability in terms of recovery coefficient (RC) and repeatability deviation (RD). This served as the foundation for the subsequent in vivo quantification. In this retrospective study, 10 patients underwent 99mTc-DPD scintigraphy, including SPECT/CT of the thorax and echocardiography examinations, as part of a clinical routine for suspected CA. The myocardial SUVmax was determined using a semi-automatic segmentation of the entire heart, excluding the descending and ascending aortas. The bone uptake was also quantified using the SUVmean parameter within the automatically delineated volume of all bones. This enabled the determination of the normalized uptake value nSUVmax, (SUVmax to SUVmean bone). Moreover, an attempt was made to apply an automatic segmentation of the myocardium based on 26% and 36% thresholds, which were developed from the torso phantom acquisitions. This approach allowed for the utilization of the injected dose (ID). Results: The planar calibration factor (CF) exceeded the volumetric cross-calibration factor (CCF) by 3.4%. The anthropomorphic phantom exhibited an underestimation of approximately 50% in the myocardium and around 21% in the kidneys. The average RD in the myocardium and kidneys was 2.3% and 1.6%, respectively. A significant quantitative separation was observed between the ATTR and control groups, comprising 6 and 4 patients, respectively (p<0.01 for SUVmax and nSUVmax, and p<0.02 for SUVmean bone). Correlation analysis revealed a weak positive correlation between SUVmax and LVMI; however, the correlation was not statistically significant (ρ = 0.31, p = 0.39). The ID in the 26% and 36% threshold-based segmented myocardium showed a relatively lower negative correlation with LVMI; although this was observed in the small ATTR cohort and was not statistically significant. The latter outcome resulted from the automatic-delineation method, which was unable to segment grade 0 and 1 patients. Conclusion: This study suggests that both CFs can be used in QSPECT. The phantom measurements indicate good repeatability and a significant quantitative underestimation, primarily due to the partial volume effect (PVE). Size and shape-specific PVE corrections, encompassing various activity concentrations of myocardium-to-blood pool ratios, are essential in QSPECT of the myocardium. Moreover, quantitative SPECT/CT utilizing 99mTc-DPD scintigraphy can effectively distinguish between patients with grade 0/1 and those with grade 2/3. This approach has the potential to enhance diagnostic accuracy and improve risk stratification.
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