Environmental Health Effects of Multiple Exposures: Systemic Risks and the Detroit River International Crossing Study

This thesis examines cumulative exposures to traffic noise and outdoor air pollution on environmental and health related quality of life in Windsor, Ontario, and provides a critical analysis of the environmental assessment process for the Detroit River International Crossing (DRIC) Study. The research utilizes a systemic risk framework to understand environmental health and stress effects of cumulative exposures. The significance of this research is based on a relative absence of literature on the systemic health risks of cumulative exposures and the need to elucidate environmental annoyance as a health outcome for risk assessment. The objectives of the research were to (1) Demonstrate the impact of high volume traffic facilities on the noise annoyance dose-response; (2) Evaluate the effect of cumulative exposures and odour annoyance on noise annoyance; (3) Conceptualize and test a model for annoyance as a health outcome of multiple exposures, and; (4) Critically appraise the capacity of environmental impact assessment to address environmental health in megaproject planning. Data from a community survey (n=610) in 2013 were combined with spatial data exposures to traffic noise and ambient nitrogen dioxide. Bivariate analyses, multivariate regression and structural equation modeling were used for the quantitative analysis. Document and media analyses were used to construct stakeholder discourses on environmental health and risk perceptions of relevance to the DRIC Study. The results of an ordinal location-scale model used to predict noise annoyance demonstrated a dose-response effect of noise, significant interactions between noise and air pollution, and a strong confounding effect of odour annoyance. A structural equation model for environmental and health related quality of life indicated that noise annoyance had a negative impact on functional mental and physical health, and that odour annoyance and levels of co-exposure were important covariates. The results of the quantitative analysis corresponded with community discourses on environmental health during the DRIC Study. Further analysis showed that the environmental assessment process obfuscated community health risks and stakeholder participation, lending support to the utilization of systemic risk perspectives and integrated environmental impact health assessments in megaproject planning. The DRIC study findings were in disagreement with public perceptions and previous research that demonstrates strong contributions of border traffic to air pollution and significant associations between air pollution and health in Windsor. The results of this thesis complement these findings by showing that ambient stressors in Windsor and in the environmental context of the DRIC megaproject had a systemic effect on health. This provides a unique contribution to the environmental health literature on cumulative effects of exposure to environmental noise and ambient pollution. It also provides a methodological contribution to systemic health risk assessment for measuring impacts of multiple environmental exposures on health related quality of life. For future research on environmental health the results warrant explicit consideration of multiple exposures and their combined effects as ambient stressors

The biological variation and fragmentation of Cardiac Myosin- Binding Protein C to diagnose acute and chronic myocardial injury

BackgroundCardiac myosin-binding protein C (cMyC) is a novel biomarker of myocardial injury, with a promising role in the triage and risk stratification of patients presenting with acute and chronic cardiac disease. To assess the suitability of cMyC for the diagnosis, monitoring and risk stratification of acute and chronic myocardial injury, we investigate (i) long- and short-term biological variation of cMyC (ii) the diagnostic performance of cMyC for suspected acute myocardial infarction in patients with renal dysfunction (iii) and the temporal release of cMyC after a brief induced ischaemia.In an attempt to overcome a major shortfall of cardiac troponin, which is low specificity towards acute myocardial infarction and the inability to differentiate between different types of myocardial injury, we study the phosphorylation and fragmentation pattern of cMyC in different types of myocardial injury in-heart and in-circulation, which includes the creation of immunoassays that would allow the quantification of intact and fragmented cMyC in the circulation from animal models and human participants with different types of myocardial injury.MethodsFor the long- and short-term biological variation studies, blood was sampled weekly and hourly from 30 and 24 healthy participants, respectively, for the determination of the biological variation, reference change value, and index of individuality. Retrospective analysis of a prospective cohort was performed to determine the diagnostic performance of cMyC compared to cardiac troponin cTn within the European Society of Cardiology ESC 0/1 h triage algorithm for the diagnosis of acute myocardial infarction, stratified by renal function. The outcome measures were safety defined by sensitivity for NSTEMI in the rule-out group; accuracy as defined by the specificity for NSTEMI in the rule-in group; NPV; PPV; and overall triage efficacy defined by the proportion of patients outside the observe zone.For the determination of the temporal release of cMyC after induced ischaemia, compared to that of cTn, biomarkers concentrations were measured at baseline and in serial blood samples from 34 consented patients who were randomly assigned to 0, 30, 60, or 90 seconds of intracoronary balloon occlusion of the left anterior descending artery. Temporal release and kinetics of the biomarkers were studied and compared.To study the phosphorylation and fragmentation pattern of cMyC in different types of myocardial injury, propofol-anesthetized swine were subjected to one of two myocardial injury protocols, model (a): 1- hour phenylephrine PE infusion for stretch-induced left ventricular (LV) stunning and cMyC release (n=5), and model (b): 1-hour Left Anterior Descending LAD occlusion for myocardial infarction MI (n=3).Myocardial tissue samples from euthanized animals at 1- and 24-h post myocardial injury were immunoblotted using high affinity anti-N-terminal cMyC antibodies and antiphosphoserine 282 antibodies.To straddle the cleavage site of cMyC and create a selective full-length FL (intact) circulating cMyC assay, high affinity anti-N-terminal and anti-C-terminal cMyC monoclonal antibodies were paired to create a sensitive electrochemiluminescence sandwich ELISA assay to complement our in-house total (all species) cMyC assay. The sensitivity of the latter was optimised to enable in-house quantification of low cMyC concentrations.Porcine serum samples from the PE model (n=3) and MI model (n=3) above, and human serum from patients with Type 1 acute myocardial infarction AMI (ST-segment elevation myocardial infarction, n=8) and Type-2 AMI (tachyarrhythmia-induced myocardial injury, n=5), were tested using both the selective FL cMyC assay and the total (all species) cMyC assay to determine the ratio of FL (intact) cMyC to total (all-species) cMyC.ResultsThe weekly biological variation, reference change value RCV and index of individuality II with 95% confidence interval (CI) were: analytical variation CVA (%) 19.5 (17.8 – 21.6), intra-individual biological variation CVI (%) 17.8 (14.8 – 21.0), inter-individual biological variation CVG (%) 66.9 (50.4 – 109.9), RCV (%) 106.7 (96.6 – 120.1)/ -51.6 (- 54.6 – -49.1) and II 0.42. There was a trend for women to have lower CVG. The calculated RCVs were comparable between genders.The hourly biological variation, reference change value RCV and index of individuality II with 95% confidence interval (CI) were: CVA (%) 11.1 (10.1-12.2), CVI (%) 13.4 (11.5- 14.2), CVG (%) 72.7 (61.6-84.7), RCV (%) 61.5 (53.0-68.4)/ -38.1 (-50.6 – -29.3) and II 0.23All long- and short-term biological variation parameters for cMyC were comparable to the respective parameters derived for cTn.On population-mean cosinor analysis of biomarker concentrations in the hourly biological variation study, cMyC and cardiac troponin T cTnT exhibited significant circadian rhythm (p = 0.015 and &lt;0.001, respectively), with 5-hours acrophase difference between cMyC and cTnT (cMyC ahead of cTnT). The impact of this physiological phenomenon on the performance of the biomarkers within unadjusted diagnostic algorithms is yet to be determined.The sensitivity of rule-out as an assessment of safety of the triage algorithm in patients with renal dysfunction was preserved using all three biomarkers, compared to patients without renal dysfunction, safety: 98.5 % [95% CI, 95.4-100] vs 96.8 % [95% CI, 94.1- 99.5], respectively, p=0.776 for cMyC; 96.9 % [95 % CI, 92.7-100] vs 97.4 % [95% CI, 95.0-99.9], respectively, p=1, for hs-TnI; and 100 % [95% CI [100-100] vs 100% [95% CI, 100-100, respectively, p=1 for hs-TnT.Accuracy of rule-in as quantified by specificity for NSTEMI of the triage algorithm in patients with renal dysfunction was lower in patients with- compared to patients withoutrenal dysfunction with all three biomarkers. Accuracy: 81.5 % [95% CI, 75.2-87.8] versus 94.5 % [95% CI, 93.0-95.9], respectively, p &lt;0.001 for cMyC; 84.2 % [95% CI,78.3- 90.1] vs 93.5 % [95% CI, 99.1-95.1], respectively, p &lt;0.001, for hs-TnI; and 86.3 % [95% CI, 80.7-91.8] vs 96.7 % [95% CI, 95.5-97.8], respectively, p &lt;0.001 for hs-TnT.Overall efficacy of the algorithm using all biomarkers was reduced, with significantly more patients allocated to the observe zone in patients with renal dysfunction compared to patients without. Overall efficacy: 57.7% [95%CI, 52.1-65.4] vs 83.0 % [95%CI, 80.7- 85.2], respectively; p &lt;0.001, for cMyC; 52.1% [95%CI, 45.3-58.8] vs 73.4% [95%CI, 70.8-76.8], respectively, p &lt;0.001 for cTnI; and 49.7% [95%CI,43.0-56.5] vs 79.2% [95%CI, 76.9-81.6], respectively, p &lt;0.001 for cTnT.Adjusting the rule-in thresholds for cMyC, could not optimise accuracy without further compromising the overall efficacy.In patients with renal dysfunction, safety, accuracy, overall efficacy and discrimination power as quantified by AUC of ROC of the 0/1 h triage algorithm for all three biomarkers were comparable.After 90 seconds of induced ischaemia, there was a significant increase at 90 minutes in the concentrations of cMyC, cTnT and cTnI, compared to baseline (p &lt;0.05). After normalizing biomarker concentrations to baseline values to allow inter-biomarker comparison, there was no significant difference between the changes of cMyC, cTnT and cTnI concentrations relative to baseline at all time points. However, there was a significantly more rapid decline in the hourly change of concentration for cMyC compared to cTnI (p=0.0312) and cTnT (p=0.048) between 3 and 4 hours after ischemia.In the study of the phosphorylation and fragmentation pattern of cMyC in different types of myocardial injury, immunoblotting showed reduced phosphorylation and increased fragmentation of cMyC in porcine myocardium subjected to ischaemic, compared to LV stretch induced, injury. There was a tendency in the latter for an increase in cMyC phosphorylation above the baseline (control) 24 hours after PE infusion.Immunoassay of cMyC in the porcine serum samples at 1-h post injury showed an increase in circulating FL (intact) cMyC in parallel with total (all species) cMyC in the PE model, whereas no detectable FL (intact) cMyC was observed in the MI model (only fragmented cMyC was present in the circulation).Immunoassay of cMyC in the human serum showed significantly higher ratio of FL (intact) to total (all species) cMyC in the tachyarrhythmia-induced myocardial injury subtype of Type 2 AMI compared to Type 1-AMI.ConclusioncMyC exhibits acceptable weekly RCV, low hourly RCV, and low weekly and hourly II, suggesting that it could be suitable for diagnosis, monitoring and risk stratification of chronic and acute myocardial injury if measured serially.In patients with renal dysfunction, the performance of cMyC-guided ESC 0/1-h triage algorithm for suspected NSTEMI is comparable to that of cardiac troponins, however, while the safety of the algorithm is high, the accuracy and overall efficacy is reduced.Induced ischemic can causes significant and comparable increase in the concentrations of cMyC and cTns, however, a faster decline in cMyC concentrations compared to cTn is observed between 3 and 4 hours after injury.Phosphorylation and fragmentation patterns of cMyC are dependent on the type of myocardial injury and might aid in the differentiation between different types of myocardial injury.</div

Causal inference methods for combining randomized trials and observational studies: a review

With increasing data availability, causal treatment effects can be evaluated across different datasets, both randomized controlled trials (RCTs) and observational studies. RCTs isolate the effect of the treatment from that of unwanted (confounding) co-occurring effects. But they may struggle with inclusion biases, and thus lack external validity. On the other hand, large observational samples are often more representative of the target population but can conflate confounding effects with the treatment of interest. In this paper, we review the growing literature on methods for causal inference on combined RCTs and observational studies, striving for the best of both worlds. We first discuss identification and estimation methods that improve generalizability of RCTs using the representativeness of observational data. Classical estimators include weighting, difference between conditional outcome models, and doubly robust estimators. We then discuss methods that combine RCTs and observational data to improve (conditional) average treatment effect estimation, handling possible unmeasured confounding in the observational data. We also connect and contrast works developed in both the potential outcomes framework and the structural causal model framework. Finally, we compare the main methods using a simulation study and real world data to analyze the effect of tranexamic acid on the mortality rate in major trauma patients. Code to implement many of the methods is provided

Heterogenous humoral and cellular immune responses with distinct trajectories post-SARS-CoV-2 infection in a population-based cohort

To better understand the development of SARS-CoV-2-specific immunity over time, a detailed evaluation of humoral and cellular responses is required. Here, we characterize anti-Spike (S) IgA and IgG in a representative population-based cohort of 431 SARS-CoV-2-infected individuals up to 217 days after diagnosis, demonstrating that 85% develop and maintain anti-S responses. In a subsample of 64 participants, we further assess anti-Nucleocapsid (N) IgG, neutralizing antibody activity, and T cell responses to Membrane (M), N, and S proteins. In contrast to S-specific antibody responses, anti-N IgG levels decline substantially over time and neutralizing activity toward Delta and Omicron variants is low to non-existent within just weeks of Wildtype SARS-CoV-2 infection. Virus-specific T cells are detectable in most participants, albeit more variable than antibody responses. Cluster analyses of the co-evolution of antibody and T cell responses within individuals identify five distinct trajectories characterized by specific immune patterns and clinical factors. These findings demonstrate the relevant heterogeneity in humoral and cellular immunity to SARS-CoV-2 while also identifying consistent patterns where antibody and T cell responses may work in a compensatory manner to provide protection

Studies on pathogenesis, clinical features and comorbidities of idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIM), a group of rare chronic inflammatory disorders, are characterized by a broad spectrum of clinical manifestations with high morbidity and mortality. The pathogenesis of IIM is largely unknown but accumulating evidence suggests that autoantibodies promote the initiation and perpetuation of the disease. The aims of this thesis were 1) to increase the understanding of the role of anti-Jo1 antibodies and the histidylt- RNA synthetase (HisRS) autoantigen in the pathogenesis of the anti-synthetase syndrome (ASS), a distinct subgroup of IIM; 2) to study the incidence and the prevalence of a lifethreatening comorbidity of IIM, the arterial and venous thrombosis, and to assess the contribution of traditional risk factors, disease characteristics and biomarkers to its occurrence. Paper I: A sensitive electro-chemiluminescence immunoassay (ECLIA) was developed to detect HisRs in serum. For the first time, HisRS was found to circulate extracellularly in the serum of healthy individuals and, with higher concentrations, in the serum of anti-Jo1- patients. Serum levels of HisRS were, instead, undetectable in anti-Jo1+ patients and correlated negatively with anti-HisRS autoantibody levels in serum. A human muscle cell culture was set up showing that primary human myoblasts could release HisRS in the culture medium with increasing amount during differentiation into myotubes and upon stimulation with insulin growth factor 1. The tolerance to endogenous HisRS was efficiently disrupted in different strains of wild-type mice by immunizing mice with murine full-length (FL) HisRS and WHEP. No obvious muscle or lung inflammation was observed in immunized mice compared to control mice. However, upon external induction of tissue-specific damage, the degree of immune engagement with consequent muscle damage and lung injury was significantly exacerbated in immunized mice compared to controls. The administration of HisRS in mice previously lung or muscle-challenged resulted in a significant reduction of the inflammation in both lungs and muscle tissue. Moreover, in vitro, HisRS inhibited the activation of T cells isolated from fresh blood of human healthy donors. Paper II and III: Total IgG were isolated from anti-Jo1+ and anti-Jo1- patients as well as from healthy controls (HC) and glycans appended to the Fc region of the IgG were explored and compared between the three groups. The Fc-glycan profile of the anti-Jo1 IgG isolated from anti-Jo1+ patients was also investigated. Total IgG and specifically anti-Jo1 IgG from IIM/ASS patients displayed a pro-inflammatory Fc-glycan profile (i.e. agalactosylation) which was overrepresented in patients with interstitial lung disease (ILD). Anti-Jo1 IgG specifically presented lower abundance of bisected and afucosylated forms and Fc-glycan characteristics correlated positively with proteins involved in inflammatory processes. IgG and IgA were isolated from serum and matching bronchoalveolar lavage fluid (BALF), collected at time of diagnosis and longitudinally, of anti-Jo1+ and anti-Jo1- patients as well as from HC to determine the reactivity levels against the FL-HisRS protein and its different constructs and splice variants. Reactivity levels of IgG and IgA isolated from BALF and serum of anti-Jo1+ patients were found to be high already at the time of diagnosis and in some cases even before diagnosis, generally decreasing thereafter. Highest reactivity was registered against the HisRS-FL and the HisRS splice variants. Moreover, IgG against HisRS-FL displayed high affinity already at the time of diagnosis. Patients with high reactivity levels towards HisRS-FL were more likely to have ILD and arthritis, but less likely to have skin rash. Noteworthy, IgG anti-WHEP reactivity in BALF correlated with poor pulmonary function. In Paper IV, the incidence of venous thromboembolic events (VTE) was assessed in patients with IIM in comparison to the general population and patient categories at high risk and the timing of risk in relation to the diagnosis of IIM were identified. In Paper V, the prevalence of arterial and venous TE was retrospectively investigated in a large cohort of IIM patients and possible traditional and/or disease-related risk factors and biomarkers linked to arterial and venous TE in patients with IIM were explored. The incidence rate of VTE was significantly higher in IIM patients than in the general population, especially during the first year after diagnosis, and remained that high even after adjusting for education level, comorbidities, cancer, treatment at baseline and competing risk of death. Among IIM patients, the risk of VTE was even more elevated in those with a history of cancer, in patients with DM, and in those with age ≥72 years. In the retrospectively assessed cohort of IIM patients, one out of 5 patients had presented with an arterial and/or venous TE at the same time of or after the diagnosis of IIM. Even though a higher frequency of male gender and essential hypertension were observed in IIM patients with reported TE and of malignancy in those with history of exclusively venous TE, only older age was an independent risk factor for TE occurrence, while autoantibodies and clinical variables did not contribute. Interestingly, lower levels of e-selectin correlated with higher odds of getting TE in IIM patients. In conclusion, the discovery of HisRS extracellularly in both healthy individuals and IIM patients supports the hypothesis that HisRS exerts other physiological functions beyond the known intracellular protein synthesis. The inhibition of T cell activation by HisRS and the impact of HisRS in reducing the degree of inflammation in mice previously immunized against HisRS and with previously induced tissue damage suggest a possible immunosuppressive activity of this protein. This could open the path for a potential new therapeutically approach in anti-Jo1 positive patients. The inflammatory Fc-glycan profile as well as the high reactivity and affinity levels in serum and BALF of anti-Jo1 antibodies (conversely undetectable serum HisRS levels) already at the time of and even before diagnosis represent new evidence supporting the role of anti-Jo1 antibodies in the pathogenesis of IIM/ASS. The high incidence and prevalence of arterial and venous thrombotic events in patients with IIM, especially close to diagnosis and in those older, male patients with essential hypertension and history of malignancy, should indicate that a proper screening and preventive measures need to be recommended in this patient population. Eselectin levels could be used as biomarkers to identify IIM patients at higher risk of presenting with TE

The role of genetic variations on gene expression and splicing in control human brain: dissection of the aetiology of complex neurological diseases

Over the past two decades there has been a realization of the importance of understanding the underlying molecular mechanism of complex neurological diseases. GWAS studies confirmed a significant association between SNPs and complex neurologic and psychiatric diseases such as Parkinson’s disease and Alzheimer’s disease. In this project, the impact of genetic variations on gene expression and alternative splicing in control post-mortem human brain tissues from twelve different regions were assessed. These are disease associated regions and support different functional roles. They are: frontal cortex, temporal cortex, occipital cortex, white matter, hippocampus, thalamus, hypothalamus, putamen, substantia nigra, medulla, cerebellum and spinal cord. Based on 1231 RNA human exon arrays, genotyped and imputed DNA samples from 137 control human brain, brain transcriptome profiles, gene and exon expression quantitative trait loci (QTL) were identified in multiple brain regions. Significant region-specific exon and gene expression QTLs were reported. Cerebellum and white matter show more unique expression profiles and expression QTLs in comparison with other brain regions. Furthermore, alternative splicing patterns were in a specific group of regions such as the cortical regions. In addition, two neurodegenerative disease related genes were investigated in detail, namely LRRK2 and MAPT. Significant regional differences in expression at mRNA and protein levels were shown. Moreover, exon QTLs correlated with the expression of specific exons located in functional protein domains of LRRK2. Also, an exon QTL has been found that shows a protective effect against Parkinson’s disease with an increase in the inclusion of exon 3 in grey matter for MAPT. This study has yielded novel regional specific expression QTLs and novel insights into the expression, regulation and function of specific genes in different regions of control human brain that are related to neurological diseases. This reference dataset is a valuable resource to complement other datasets for research into the complex genetics of neurological diseases

Human Herpesvirus 8 Seropositivity Among Sexually Active Adults in Uganda

Sexual transmission of human herpesvirus 8 (HHV8) has been implicated among homosexual men, but the evidence for sexual transmission among heterosexual individuals is controversial. We investigated the role of sexual transmission of HHV8 in a nationally representative sample in Uganda, where HHV8 infection is endemic and transmitted mostly during childhood.The study population was a subset of participants (n = 2681) from a population-based HIV/AIDS serobehavioral survey of adults aged 15-59 years conducted in 2004/2005. High risk for sexual transmission was assessed by questionnaire and serological testing for HIV and herpes simplex virus 2. Anti-HHV8 antibodies were measured using two enzyme immunoassays targeting synthetic peptides from the K8.1 and orf65 viral genes. The current study was restricted to 2288 sexually active adults. ORs and 95% CIs for HHV8 seropositivity were estimated by fitting logistic regression models with a random intercept using MPLUS and SAS software.The weighted prevalence of HHV8 seropositivity was 56.2%, based on 1302 seropositive individuals, and it increased significantly with age (P(trend)<0.0001). In analyses adjusting for age, sex, geography, education, and HIV status, HHV8 seropositivity was positively associated with reporting two versus one marital union (OR:1.52, 95% CI: 1.17-1.97) and each unit increase in the number of children born (OR: 1.04, 95% CI: 1.00-1.08), and was inversely associated with ever having used a condom (OR: 0.64, 95% CI: 0.45-0.89). HHV8 seropositivity was not associated with HIV (P = 0.660) or with herpes simplex virus 2 (P = 0.732) seropositivity. Other sexual variables, including lifetime number of sexual partners or having had at least one sexually transmitted disease, and socioeconomic variables were unrelated to HHV8 seropositivity.Our findings are compatible with the conclusion that sexual transmission of HHV8 in Uganda, if it occurs, is weak

Phage display based approach for identifying novel HDL particle binders: Recombinant HDL antibodies as novel diagnostic tool for risk assessment and monitoring of coronary artery disease

Cholesterol content of high density lipoprotein (HDL-C) has been a well established cardiac risk marker for coronary artery disease (CAD), a major cause of mortality worldwide. Various epidemiological and experimental animal studies have shown the inverse correlation between concentration of HDL-C and CAD. However, recent pharmacological studies and genetic studies have demonstrated lack of protection from CAD despite of elevated HDL-C. HDLs are a heterogenous group of particles with varying protein/lipid composition and anti-atherogenic (atheroprotective) function. Atheroprotective HDL particles could turn dysfunctional and thereby attenuate their atheroprotective properties under particular circumstances such as among subjects with metabolic syndrome. Therefore, it is hypothesised that risk assessment and clinical management of atherosclerotic CAD (ATCAD) should target these dysfunctional HDL particles rather than focusing on HDL-C. The aim of this thesis was to develop antibodies against HDL particles by using phage display based antibody library and implement them for development of immunoassay for diagnosis of CAD and its risk estimation in symptomatic and asymptomatic individuals. To that end in publication I, a large set of HDL antibodies were isolated against the HDL derived from CAD patients. These antibodies were characterized against different HDL preparations, most abundant proteins of HDL (i.e. apoA-I and apoA-II) and plasma. A variety of binders were identified. In publication II, three distinct pairs of HDL antibodies which were mainly recognizing the apoA-I were employed to develop three different phage based two-site apoA-I immunoassays identified as assay 022-454, assay 109-121 and assay 110-525. Assays 109-121 and 110-525 showed promise to further improve the diagnostic and predictive value for cardiac conditions. In publication III, simplified versions of two of the phage based two-site immunoassays (109-121 and 110-525) is presented and clinically assessed with a cohort of cardiac patients. Higher level of apoA-I measured with the assay 110-525 showed clear association with ATCAD especially in patients not using lipid lowering medication. In light of these observations, it can be said that that a large number of antibodies were obtained against the intact HDL molecules with phage display approach. The results from testing clinical specimens with the new combinations of HDL binders suggest a different and improved performance over existing test technologies in estimating risk of ATCAD However, further evaluation of these HDL binders with larger cohorts is clearly warranted. In addition, these antibodies could also serve as analytical tools for unravelling the etiology and the pathological process of atherosclerosis in relation to HDL.Uudenlaisten HDL-partikkelia tunnistavien sitojien kehittäminen faaginäyttötekniikan avulla: rekombinanttiset HDL-vasta-aineet uudenlaisina diagnostisina työkaluina sepelvaltimotaudin riskinarvioinnissa ja seurannassa Sepelvaltimotauti on merkittävä kuolinsyy maailmalaajuisesti. Suuritiheyksisen lipoproteiinin kolesterolipitoisuus (high density lipoprotein cholesterol, HDL-C) on laajalti tunnustettu sepelvaltimotaudin riskitekijä. Monet epidemiologiset sekä kokeelliset eläintutkimukset ovat osoittaneet, että HDL-C on käänteisesti yhteydessä sepelvaltimotautiin. Viimeaikaisissa farmakologisissa ja geneettisissä tutkimuksissa ei ole kuitenkaan pystytty osoittamaan, että koholla oleva HDL-C suojelisi sepelvaltimotaudilta. HDL-partikkelit ovat heterogeeninen ryhmä partikkeleja, joiden proteiini- ja lipidikoostumus sekä ateroskleroosilta suojelevat ominaisuudet vaihtelevat. Tietyissä yhteyksissä, kuten esimerkiksi metabolista oireyhtymää sairastavilla, ateroskleroosilta suojaavat HDL-partikkelit voivat muuntua toiminnaltaan, jolloin niiden ateroskleroosilta suojaavat ominaisuudet voivat heikentyä. Tästä syystä on esitetty, että ateroskleroosiin liittyvän sepelvaltimotaudin riskinarvio ja hoito pitäisi perustua toiminnaltaan heikentyneisiin HDL-partikkeleihin HDL-C:n sijaan. Tämän väitöskirjan tavoitteena oli löytää vasta-aineita HDL-partikkeleille faaginäyttötekniikkaan perustuvalla vasta-ainekirjastolla sekä kehittää niitä käyttäen immunomäärityksiä sepelvaltimotaudin diagnostiikkaan ja riskinarviointiin. Näiden tavoitteiden puitteissa julkaisussa I eristettiin suuri ryhmä HDL-vasta-aineita käyttämällä kohteena sepelvaltimotautipotilaiden HDL-partikkeleita. Vasta-aineet karakterisoitiin käyttämällä hyväksi erilaisia HDL-valmisteita, HDL-partikkelien yleisimpiä proteiineja (apoA-I ja apoA-II) ja plasmaa. Eristettyjen vasta-aineiden joukosta tunnistettiin erilaisia vasta-aineita. Julkaisussa II kehitettiin kolme apoA-I-proteiinia tunnistavaa kaksipuolista immunomääritystä (määritys 022-454, määritys 109-121 ja määritys 110-525), joissa hyödynnettiin kolmea keskenään erilaista, pääosin apoA-I -proteiinia tunnistavaa vastaaineparia. Määritykset 109-121 ja 110-525 näyttivät lupaavilta sen suhteen, että ne voisivat pystyä parantamaan sydänsairauksia diagnostiikkaa ja ennustettavuutta. Käsikirjoituksessa III esitetään yksinkertaistetut versiot kahdesta faagipohjaisesta kaksipuolisesta immunomäärityksestä (109-122 ja 110-525) ja arvioidaan niiden kliininen toiminta sydänpotilasryhmän kanssa. Määrityksellä 110-525 mitattu korkeampi apoA-I pitoisuus oli selvästi yhteydessä ateroskleroosiin liittyvään sepelvaltimotautiin erityisesti niillä potilailla, joilla ei ollut lipidejä laskevaa lääkitystä. Näiden havaintojen valossa voidaan todeta, että tutkimuksessa löydettiin faaginäyttötekniikalla useita HDL-molekyylejä tunnistavia vastaaineita. Kliinisillä näytteillä saatujen tulosten perusteella näiden uusien sitojien kombinaatiot näyttävät ennustavan ateroskleroosiin liittyvää sepelvaltimotautia eri tavalla ja paremmin kuin nykyään käytössä olevat testimenetelmät. Tulokset pitää kuitenkin vahvistaa laajemmilla potilasjoukoilla. Tutkimuksessa löydetyt vasta-aineet voisivat lisäksi olla hyödyllisiä analyyttisinä työkaluina tutkittaessa ateroskleroosin etiologiaa ja patologisia prosesseja suhteessa HDL-partikkeleihin

The gut microbiota and metabolome are associated with diminished COVID-19 vaccine-induced antibody responses in immunosuppressed inflammatory bowel disease patients

Background: Patients with inflammatory bowel disease (IBD) treated with anti-TNF therapy exhibit attenuated humoral immune responses to vaccination against SARS-CoV-2. The gut microbiota and its functional metabolic output, which are perturbed in IBD, play an important role in shaping host immune responses. We explored whether the gut microbiota and metabolome could explain variation in anti-SARS-CoV-2 vaccination responses in immunosuppressed IBD patients. Methods: Faecal and serum samples were prospectively collected from infliximab-treated patients with IBD in the CLARITY-IBD study undergoing vaccination against SARS-CoV-2. Antibody responses were measured following two doses of either ChAdOx1 nCoV-19 or BNT162b2 vaccine. Patients were classified as having responses above or below the geometric mean of the wider CLARITY-IBD cohort. 16S rRNA gene amplicon sequencing, nuclear magnetic resonance (NMR) spectroscopy and bile acid profiling with ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS) were performed on faecal samples. Univariate, multivariable and correlation analyses were performed to determine gut microbial and metabolomic predictors of response to vaccination. Findings: Forty-three infliximab-treated patients with IBD were recruited (30 Crohn's disease, 12 ulcerative colitis, 1 IBD-unclassified; 26 with concomitant thiopurine therapy). Eight patients had evidence of prior SARS-CoV-2 infection. Seventeen patients (39.5%) had a serological response below the geometric mean. Gut microbiota diversity was lower in below average responders (p = 0.037). Bilophila abundance was associated with better serological response, while Streptococcus was associated with poorer response. The faecal metabolome was distinct between above and below average responders (OPLS-DA R2X 0.25, R2Y 0.26, Q2 0.15; CV-ANOVA p = 0.038). Trimethylamine, isobutyrate and omega-muricholic acid were associated with better response, while succinate, phenylalanine, taurolithocholate and taurodeoxycholate were associated with poorer response. Interpretation: Our data suggest that there is an association between the gut microbiota and variable serological response to vaccination against SARS-CoV-2 in immunocompromised patients. Microbial metabolites including trimethylamine may be important in mitigating anti-TNF-induced attenuation of the immune response. Funding: JLA is the recipient of an NIHR Academic Clinical Lectureship (CL-2019-21-502), funded by Imperial College London and The Joyce and Norman Freed Charitable Trust. BHM is the recipient of an NIHR Academic Clinical Lectureship (CL-2019-21-002). The Division of Digestive Diseases at Imperial College London receives financial and infrastructure support from the NIHR Imperial Biomedical Research Centre (BRC) based at Imperial College Healthcare NHS Trust and Imperial College London. Metabolomics studies were performed at the MRC-NIHR National Phenome Centre at Imperial College London; this work was supported by the Medical Research Council (MRC), the National Institute of Health Research (NIHR) (grant number MC_PC_12025) and infrastructure support was provided by the NIHR Imperial Biomedical Research Centre (BRC). The NIHR Exeter Clinical Research Facility is a partnership between the University of Exeter Medical School College of Medicine and Health, and Royal Devon and Exeter NHS Foundation Trust. This project is supported by the National Institute for Health Research (NIHR) Exeter Clinical Research Facility. The views expressed are those of the authors and not necessarily those of the NIHR or the UK Department of Health and Social Care