| 1. |
- Byström, Sanna
(författare)
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Affinity assays for profiling disease-associated proteins in human plasma
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer.The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided.Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD.In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies.
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| 2. |
- Dodig-Crnković, Tea
(författare)
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On the application and validation of multiplexed affinity assays
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are essential macromolecules that carry out complex functions in human cells, tissues, and organs. They regulate a diverse set of biological processes and protect against pathogens. However, dysregulation or malformation of proteins can cause disease. By characterizing proteins in health and disease, we can gain insights into disease aetiology and identify druggable targets to treat disorders. By bringing protein discoveries from the research lab into clinical practice, protein assays have been and will continue to be important tools for enabling and improving medical decision-making. The work presented in this thesis concerns both exploratory and targeted affinity-based assays applied for the study of proteins. High-throughput and multiplexed suspension bead arrays have been the primary technology for measuring proteins with antibodies in samples such as human blood. Identification and validation of protein-protein interactions that may provide novel insights into the druggable proteome have also been carried out. Throughout the projects, methods for validating the observations have been pursued and include replication in independent sample sets, as well as the assessment of antibody selectivity via other proteomics assays or orthogonal methods such as genetic associations. In Paper I, we used multiplexed exploratory antibody arrays comprising almost 1.500 affinity binders to study proteins that circulate in plasma. Here, the focus was to determine the longitudinal variability of proteins. We analysed samples from 101 clinically healthy individuals, collected each third month for one year. The protein data provided insights into inter-individual diversity and the unique molecular fingerprint of each participant. We found that 49% of the studied proteins were stable across one year, as these had low variability in each individual. Eight modules, each containing 11-242 proteins, were found to co-vary across one year. We also found genetic variations to influence 15 of the detected protein profiles and confirmed selected indications in an independent set of 3.000 subjects. In summary, we observed the existence of individual-specific protein profiles and found that short-term and continuous changes occurred in almost every participant. In Paper II, we investigated blood-derived serum and plasma to identify age-associated proteins. We started from a large set of exploratory antibody bead arrays to screen 156 individuals aged 50-92 years. We found protein profiles of the histidine-rich glycoprotein (HRG) to be significantly associated with age. This association was further corroborated by the analysis of >4.000 individuals from eight additional and independent sets of blood samples. We further validated the HRG protein profiles by sandwich assays and protein microarrays developed in-house. Comparing genetic data and HRG profiles obtained by two independent antibodies, we observed strong but inverse associations to the genetic variants for two anti-HRG antibodies. In Paper III, we applied multiplexed assays for the detection of autoantibodies against cancer-testis antigens (CTAs) in 133 non-small cell lung cancer (NSCLC) patients. We found reactivity against 29 unique CTAs exclusively in cases, compared to 57 matched controls with benign lung diseases. The presence of six CTAs was further confirmed in an independent set of 34 NSCLC cases. Analysis of longitudinal samples from seven patients demonstrated that the presence of CTA autoantibodies was stable over time for each of the individuals. In Paper IV, we developed a novel multiplexed sandwich-immunoassay for the detection of interaction partners to G-protein coupled receptors (GPCRs). This pharmaceutically important family of membrane proteins is believed to be regulated by another group of receptor activity-modulating proteins (RAMPs) by the formation of protein complexes. We studied cell lysates expressing combinations of 23 GPCRs with three RAMPs. We confirmed most of the previously reported interaction pairs and additionally found evidence for 15 new GPCR-RAMP complexes. All interactions were validated using epitope tags that were engineered onto the proteins. Selected complexes were further validated by in situ proximity ligation assays performed in cell membranes. In summary, the work included in this thesis describes the use of multiplexed affinity-based assays for research within plasma proteomics and the interrogation of protein complexes. The work highlights the method’s potential for the identification of circulating proteins that may aid and add to the current knowledge about human health and disease.
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| 3. |
- Holmberg, Benny, 1958-
(författare)
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Analysis of risk factors in patients with severe chronic kidney disease. The role of atorvastatin.
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background and aim: There had been no randomized end-point studies with statins for patients with severe renal failure. The purpose of this prospective, open, randomized, controlled study was to investigate whether atorvastatin (10 mg/day) would alter cardiovascular end-points and the overall mortality rate of patients with chronic kidney disease stage 4 or 5 (creatinine clearanceMaterial & Methods: This was an open, prospective, randomized study. A total of 143 patients were included: 73 were controls and 70 were prescribed 10 mg/day of atorvastatin. As efficacy variables, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglyceride levels were determined at the start of the study and at 1, 3, 6, 12, 18, 24, 30 and 36 months. The primary end-points were all cause of mortality, non-lethal acute myocardial infarction, and coronary artery intervention. Various risk factors were studied. In the 97 patients on haemodialysis inter dialysis weight gain (IDWG) was calculated as ultrafiltration in kg/body weight in kg given in percentage of the weight. The burden of IDWG was analyzed.Results: In the atorvastatin group, total cholesterol and low-density lipoprotein cholesterol were significantly reduced, the latter by 35% at 1 month and then sustained. Atorvastatin was withdrawn in 23% of patients due to unacceptable side effects, most frequent complaints being gastrointestinal discomfort and headache. Primary end-points occurred in 74% of the subjects. There was no difference in cardiovascular endpoint and survival between the control and atorvastatin groups. The 5-year end-point-free survival rate from study entry was 20%. There was no evidence of more benefit of atorvastatin for patients with diabetes mellitus and chronic kidney disease versus the other patients; instead plasma fibrinogen increased. The IDWG was significantly larger in patients who suffered from end-points due to cardiovascular reasons, cardiac reasons, congestive heart failure, aortic aneurysm, and intracerebral bleeding.Conclusion: These data showed that in contrast to other patient groups, patients with severe chronic kidney disease 4 and 5, including those with diabetes mellitus, seem to have no benefit from 10mg/day of atorvastatin. Instead we found a high IDWG to be an important risk factor that should be prevented. There was no evident connection between atorvastatin medication and IDWG.
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| 4. |
- Holmgren, Klas, 1989-
(författare)
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Permanent stoma after anterior resection for rectal cancer : prevalence and mechanisms
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- While sphincter-saving surgery constitutes standard treatment for rectal cancer, anterior resection still harbours a significant risk of a permanent stoma in the long run. Although anastomotic leakage plays a major role in this surgical dilemma, the exact mechanisms are not known, while surveys indicate a stoma-free outcome is essential for a majority of patients. To address this issue, the overall aim of the present thesis was to investigate the permanent stoma prevalence in patients undergoing anterior resection for rectal cancer in Sweden, and to identify plausible mechanisms that impede prospects of a stoma-free outcome.In a population-based cohort, chart review of patients who had anterior resection for rectal cancer in the Northern healthcare region in Sweden between 2007 and 2013 showed that 75 out of 316 (24%) patients ended up with a permanent stoma. Of 274 patients (87%) primarily defunctioned with a stoma, 229 underwent stoma closure, 21 (9%) of whom suffered major complications that required return to theatre or worse. A permanent stoma was shown to be more common among patients with anastomotic leakage and an advanced tumour stage.A registry-based method to estimate nationwide stoma outcome after anterior resection for rectal cancer was developed, using data from the Swedish Colorectal Cancer Registry and the National Patient Registry. With a chart-reviewed cohort as reference, stoma outcome was assessed with a positive predictive value of 85.1%, and a negative predictive value of 100.0%. In patients operated in Sweden between 2007 and 2013, the registry-based method determined that 942 out of 4768 (19.8%) had a permanent stoma, while stoma rates varied substantially between different healthcare regions.In a 1:1 matched case-control study of 82 patients who had curative resection for non-disseminated colorectal cancer, a subgroup analysis of 34 patients with rectal cancer displayed biomarker aberrations in serum measured preoperatively in those with anastomotic leakage. Compared to complication-free controls, 15 proteins related to inflammation were elevated, of which two (C-X-C motif chemokine 6, and C-C motif chemokine 11) remained significant after adjustment for multiple testing.Based on a cohort of 4529 patients who had anterior resection, tumour height served as a proxy to determine the extent of mesorectal excision, while long-term stoma outcome was classified using a previously validated registry-based method. Defunctioning stomas significantly decreased chances of a stoma-free outcome, especially in patients undergoing partial mesorectal excision; for these patients, faecal diversion was also least beneficial in terms of reducing anastomotic leakage.In conclusion, every fifth patient undergoing anterior resection for rectal cancer in Sweden eventually ends up with a permanent stoma. Although construction of a defunctioning stoma decreases the risk of symptomatic anastomotic leakage, subsequent takedown surgery carries a substantial risk of major complications, while chances of a long-term stoma-free outcome become significantly reduced. To facilitate selective use of faecal diversion, novel markers to identify high-risk anastomoses prior to surgery have been identified, but require validation in larger prospective settings. Anterior resection without a defunctioning stoma should be considered in appropriately informed patients for whom a stoma-free outcome is of importance. In particular, this holds true for patients eligible for partial mesorectal excision, where anastomotic dehiscence is less frequent and the advantageous effects of a defunctioning stoma are limited.
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| 5. |
- Häggmark, Anna, 1985-
(författare)
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Neuroproteomic profiling of human body fluids
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis provides results from affinity based studies where human body fluids were profiled to find markers for neurological diseases. Both proteins and autoantibodies were analysed using microarray technologies that can profile hundreds of analytes and hundreds of samples in parallel using small sample volumes. A central element in this work was to develop and apply new methods to study cerebrospinal fluid (CSF), which is the fluid in direct contact with the brain. CSF contains proteins reflecting the physiological state of the central nervous system and therefore offers a unique insight into proteins associated to neurological disorders. As a complement to CSF, bloodderived samples such as serum and plasma, were also investigated as these represent potential sources of disease related proteins. The work presented here summarises the development of assay protocols to study protein and autoantibodies in CSF and blood using planar and bead-based microarrays.In Paper I, an antibody-based protocol was developed to enable multiplexed protein profiling in CSF. The protocol was then applied for a first analysis within multiple sclerosis (MS) patients. In Paper II, the results were further evaluated in additional CSF as well as plasma samples. Based on the CSF analysis we found two proteins associated to MS; GAP43, a protein related to disease progression and SERPINA3, a protein involved in inflammation. In addition, four other proteins; IRF8, METTL14, IL7 and SLC30A7, were found to have altered plasma levels between the patient groups. The expression of these proteins were further investigated by immunofluorescent staining of human brain tissue, revealing differential localisation of proteins in diseased and healthy brain. In Paper III, a study on extensive protein profiling of plasma in the context of another neurodegenerative disorder, amyotrophic lateral sclerosis (ALS), is described. The levels of three proteins, namely NEFM, RGS18 and SCL25A20, were found to be elevated in ALS patients compared to controls. Among these, NEFM also indicated association to disease subtype as the levels were elevated in patients with definite compared to suspected diagnosis.In addition to antibodies, we also utilised antigens on microarrays to screen for the presence of autoantibodies in body fluids. In Paper IV, a strategy for this analysis was developed using protein fragments and two types of microarrays. This strategy was then applied for profiling of the autoantibody repertoire of MS patients, revealing 51 protein fragments with potential disease relevance. Interestingly, comparison of plasma and CSF samples obtained from the same patients indicated high concordance of antibodies between the two body fluids. In Paper V, a similar strategy was applied to narcolepsy, another neurological disorder. Our investigation of antibodies in serum revealed higher reactivity towards METTL22, NT5C1A and TMEM134 compared to controls in two independent sample materials.In conclusion, the presented work constitutes a framework of proteomic assays for enhanced exploration of proteins and autoantibodies in neuroscience. Moreover, we have reported identification of several potential disease markers to be further investigated within neurological disorders.
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| 6. |
- Just, David, 1987-
(författare)
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On the profiling of autoantibodies in psychiatric disorders
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There is a great need to increase our understanding of diseases affecting the brain and their underlying pathogenic mechanisms. To address this need, the work presented in this thesis applied affinity based proteomic techniques to profile proteins, and to investigate protein profiles and the autoantibody repertoire in brain related disorders. Studies included in this thesis cover traumatic brain injuries, first-episode psychosis, schizophrenia and obsessive-compulsive disorders. Paper I describes the profiling of rat serum samples of a traumatic brain injury model to increase the understanding of injury related protein markers and their potential role in patient outcome. Changes in protein profiles over time were characterized as well as potential injury markers related to oxygen intake. Paper II-IV describe the use of protein fragment-based arrays to investigate potential pathogenic autoantibodies associated to the disease. In Paper II possible predictive autoantibodies for the development of schizophrenia were identified, Paper III identified probable brain reactive autoantibodies in schizophrenia patients and Paper IV described the exploration of autoantibodies which might have an association to obsessive-compulsive disorder. Further characterization of these autoantibody repertoires in psychiatric disorders and future efforts could increase our understanding of their role in the associated diseases. Taken together, this work provides the basis for future research in the search for novel disease associated proteins and autoantibody profiles in brain related disorders. An increased understanding and additional diagnostic or prognostic markers of these disorders would be beneficial for both researchers and patients.
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| 7. |
- 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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| 8. |
- Qundos, Ulrika, 1983-
(författare)
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Antibody based plasma protein profiling
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided.Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V).As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected.
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| 9. |
- Zandian, Arash, 1988-
(författare)
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Array-based Autoantibody Profiling and Epitope Mapping
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibodies are a class of proteins that are made by the immune system to recognize harmful organisms and molecules. Their exceptional capability of specifically recognizing molecules has been investigated for over a century and information thereof has been utilized for a variety of applications including vaccine and generation of therapeutic antibodies. Occasionally, instead of protecting the host against pathogens, antibodies can recognize constituents of the host and thereby cause an autoimmune reaction that eventually can lead to a disease. Therefore, it is of great interest to understand what the antibodies bind to and their specificities. The last decades of technical development and availability of protein and peptide microarrays have enabled large-scale profiling of antibodies and precise determination of their specificities through epitope mapping. In this thesis the aim was to use affinity proteomics tools to profile antibodies, determine their specificities, and discover potential associations of autoantigens to disease by analyzing blood-derived samples with microarray-based methods. In Paper I, 57 serum samples from patients with the suggested autoimmune disease narcolepsy, were analyzed on planar antigen microarrays with 10,846 human protein fragments. Verification on an independent sample collection consisting of serum samples from 176 individuals, revealed METTL22 and NT5C1A as two potential autoantigens. In Paper II, antibodies from 53 plasma samples from patients with first-episode psychosis, a condition suggested to have a partial autoimmune component, were analyzed on planar antigen microarrays with 2,304 human protein fragments. After a follow-up study of the patients, antibodies toward an antigen representing the three proteins, PAGE2, PAGE2B, PAGE5, was found associated to an increased risk of developing schizophrenia. In Paper III, serum and plasma samples from patients with the autoimmune diseases multiple sclerosis and narcolepsy, were epitope mapped on high-density peptide microarrays with approximately 2.2 million peptides. Technical and biological verification, by using other microarray technology and analyzing samples from 448 patients, revealed one peptide for multiple sclerosis and narcolepsy, representing the proteins MAP3K7 and NRXN1, with higher antibody reactivity towards in each group, respectively. In Paper IV, purified polyclonal antibodies raised against a surface antigen found on malaria-infected erythrocytes, were profiled on the peptide microarrays representing all proteins found on malaria-infected erythrocytes derived from Plasmodium falciparum. Then, different Plasmodium falciparum strains were analyzed by immunofluorescence microscopy and western blots, using the epitope mapped antibodies. The performance of the immunoassays were compared to the identified epitopes, and validated by RNA sequencing. In conclusion, these investigations describe multiplex methods to identify and characterize antibodies, their disease association and epitopes. Follow-up studies are needed to determine their potential use and clinical value.
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