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Yes / Context: Urinary biomarkers are promising as simple alternatives to cystoscopy for the diagnosis of de novo and recurrent bladder cancer. Objective: To identify a highly sensitive and specific biomarker candidate set with potential clinical utility in bladder cancer. Materials and methods: Urinary biomarkers concentrations were determined by ELISA. The performance of individual markers and marker combinations was assessed using ROC analysis. Results: A 5-biomarker panel (IL8, MMP9, VEGFA, PTGS2 and EN2) was defined from the candidate set. Discussion and conclusion: This panel showed a better overall performance than the best individual marker. Further validation studies are needed to evaluate its clinical utility in bladder cancer. / This work has been supported in part by the European Commission Program DIPROMON - HEALTH-F5-2012-306157-2: Development of an integrated protein- and cell-based device for non-invasive diagnostics in the urogenital tract.

Background: This thesis focuses on the incidence and remission of reported food hypersensitivity in schoolchildren followed from 8 to 12 -years of age and the prevalence of hypersensitivity to milk, egg, cod and wheat among 12-year olds investigated by reported data, clinical investigation and double-blind placebo-controlled food challenges and biomarkers. Methods: The studies are mainly based on a population based cohort recruited in 2006 from three municipalities in Northern Sweden. All children in first and second grade, aged 7-8 years, were invited to a parental questionnaire study and 2585 (96% of invited) participated. The children in two of the municipalities were also invited to a skin prick test with airborne allergens. At age 11-12 years, there was a follow-up of the cohort using the same methods, with the addition of a child interview and assessment of body mass index (BMI). At the follow-up, children who reported milk hypersensitivity were invited to structured interviews and children reporting complete elimination of milk, egg, cod or wheat due to perceived hypersensitivity were invited to a clinical examination and blood sampling. According to test results, the children were categorized into different food hypersensitivity phenotypes according to preset criteria. Children categorized as current food allergy were then invited to further evaluation with a double-blind placebo-controlled food-challenge using newly developed recipes. Before their use, the recipes were successfully validated regarding detectable sensorial differences between the active and placebo substances in a separate cohort of healthy schoolchildren (n=275). Before and after the challenge series blood samples were collected for analyses of cytokine mRNA expression in peripheral blood mononuclear cells including hallmark cytokines for the humoral allergy-promoting T helper (Th) 2 response, cellular cytotoxicity-promoting Th1 response, inflammatory-, and T regulatory responses. Fecal inflammatory biomarkers were also analyzed before and after the challenge series. Results: Reported food hypersensitivity increased from 21% at age 7-8 years to 26% at 11-12 years. There was a high incidence (15%) as well as a high remission (33%) of reported food hypersensitivity. Risk factors associated with incidence and remission were different for milk hypersensitivity and hypersensitivity to foods other than milk. The agreement between reported symptoms to milk, egg, cod, wheat, soy and peanut and sensitization to the culprit food was poor. At 11 to 12-years of age the prevalence of reported allergy to milk, egg, cod or wheat was 4.8% while the allergy prevalence according to clinical evaluation was 1.4%. This figure was further halved when evaluated with double-blind placebo-controlled food challenges. The majority of children with reported allergy to milk, egg, cod and wheat were categorized as other food hypersensitivity phenotypes, the most common being probable lactose intolerance (40%) and outgrown food allergy (19%). Even though reported milk hypersensitivity among the 11-12 year olds was 14.5%, only 3% were categorized as current milk allergy. Current and outgrown milk allergy was associated with other atopic disorders and a lower BMI (OR 0.8, 95% CI 0.80-0.98). Before the challenge series, the mRNA expression of the cytokines IL-13 and IL-10 were higher among children with a positive compared to a negative challenge outcome. Conclusion: Reported food hypersensitivity was common among school children in Northern Sweden and increased from 7-8 years to 11-12 years of age, and both the incidence and remission of reported hypersensitivity was high. There was an 8-fold difference in the prevalence of allergy to milk, egg, cod or wheat when reported data was assessed by clinical examinations and double-blind placebo-controlled food challenges. Allergy to milk, egg, cod and wheat was an uncommon cause of complete avoidance of these foods due to perceived hypersensitivity. Some of the analyzed biomarkers might serve as prognostic markers for symptomatic, IgE-mediated food allergy but need further validation. / Bakgrund: Födoämnesöverkänslighet håller på att bli ett stort och kostsamt hälsoproblem i västvärlden. Prevalensen av rapporterad födoämnes-överkänslighet bland barn ökar, men det är fortfarande oklart om detta avspeglar en sann ökning i populationen. Det finns en stor spridning mellan uppmätta prevalenstal i olika studier och i de få studier där man följt upp rapporterade data med objektiva metoder ses en hög överrapportering. Data saknas om reell prevalens av födoämnesöverkänslighet bland skolbarn i Sverige. Befintliga prevalensdata baseras på rapporterad födoämnes-överkänslighet och studier saknas där angiven födoämnesöverkänslighet i en barnpopulationskohort validerats med objektiva metoder. Även om dubbelblinda provokationer räknas som ”gold standard” används i praktiken endast sjukhistoria, pricktest och analys av specifikt Immunoglubulin E (IgE) samt öppna provokationer för diagnostik. Metoderna har flera felkällor och mer tillförlitlig diagnostisk behövs, särskilt vid sena och svårtolkade symtom. Korrekt diagnos är särskilt viktig när baslivsmedel har eliminerats eftersom kostrestriktionerna riskerar att leda till negativa konsekvenser för livskvalitet och intag av viktiga näringsämnen. Syfte: Studierna i denna avhandling fokuserade på incidens och remission av födoämnesöverkänslighet bland skolbarn i Norrbotten, som följdes från 7-8 år till 11-12 års ålder, och på prevalens av överkänslighet mot mjölk, ägg, fisk och vete bland 12-åringar, undersökt med rapporterade data, klinisk undersökning samt dubbelblinda placebokontrollerade födoämnes-provokationer och biomarkörer. Metod: Den huvudsakliga delen av det här projektet är baserat på en populations-baserad kohort som rekryterades 2006 från 3 kommuner i norra Sverige. Föräldrar till alla barn i klass 1 och 2 (7-8 år) bjöds in till ett frågeformulär, som besvarades av 96% (n=2585) av de inbjudna. Barnen från två av kommunerna, Luleå och Kiruna, bjöds också in till ett pricktest med 10 vanliga luftburna allergen och 90% (n=1700) av de inbjudna deltog. År 2010, när barnen var 11-12 år, gjordes en studieuppföljning med samma metoder och med ytterligare tillägg av en intervju med barnet och bestämning av body mass index (BMI). Studiedeltagandet i enkäter och pricktest var lika högt vid uppföljningen som vid studiestart. Vid studieuppföljningen bjöds barn med rapporterad mjölköverkänslighet in till en strukturerad intervju och barn som helt undvek mjölk, ägg, fisk eller vete på grund av upplevd överkänslighet, bjöds in till klinisk undersökning och provtagning. Baserat på testresultaten kategoriserades barnen i olika fenotyper av födoämnesöverkänslighet utifrån förutbestämda kriterier. Barn som bedömdes ha en aktuell födoämnesallergi bjöds därefter in till vidare utredning med dubbelblind placebokontrollerad födoämnes-provokation. De recept som användes vid de dubbelblinda provokationerna hade dessförinnan validerats avseende detekterbara smak- och konsistens-skillnader mellan aktiv- och placebosubstans i en separat kohort av friska skolbarn (n=275). Före och efter den dubbelblinda provokationen samlades blodprover in för analys av cytokin mRNA-uttryck i mononukleära celler. Analyserna inkluderade cytokiner kännetecknande för humoralt allergidrivande T-hjälpar 2 (Th2) svar, cellulärt cytotoxiskt drivande Th1 svar samt inflammatoriskt- och T-reglerande svar. Vidare insamlades avföringsprover för analys av inflammatoriska biomarkörer före och efter genomgången provokationsserie. Resultat: Prevalensen av föräldrarapporterad födoämnesöverkänslighet ökade från 21% vid 7-8 år till nästan 26% vid 11-12 års ålder. Incidensen av rapporterad födoämnesöverkänslighet var hög (15%), liksom remissionen (33%). Riskfaktorer associerade med incidens och remission var olika för mjölk-överkänslighet och överkänslighet mot andra födoämnen. Vi såg också en bristande samstämmighet mellan föräldrarapporterad överkänslighet mot mjölk, ägg, fisk, vete, soja och jordnöt och IgE-sensibilisering mot det aktuella födoämnet. Vid 11-12 års ålder var prevalensen av rapporterad allergi mot mjölk, ägg, fisk eller vete 4.8%, medan prevalensen baserad på klinisk undersökning och provtagning var 1.4%. Prevalenssiffran halverades ytterligare när kliniskt bedömd födoämnesallergi validerades med dubbelblinda placebo-kontrollerade födoämnesprovokationer. Majoriteten av barnen med rapporterad allergi mot mjölk, ägg, fisk eller vete klassificerades som andra fenotyper av födoämnesöverkänslighet, varav de vanligast förekommande var möjlig laktosintolerans (40%) och utläkt födoämnesallergi (19%). Även om förekomsten av rapporterad mjölköverkänslighet bland 11-12 åringarna var så hög som 14.5%, kategoriserades bara 3% av dessa som en aktuell mjölkallergi. Mjölkallergi, aktuell eller utläkt, var associerat med andra atopirelaterade tillstånd och ett lägre BMI (OR 0.82, 95% CI 0.80-0.98) jämfört med barn som inte undvek mjölkprodukter. Före den dubbelblinda provokationsserien var mRNA-uttrycket av den Th2-relaterade cytokinen IL-13 och den regulatoriska cytokinen IL-10 högre bland barn med provokationspåvisad födoämnesallergi jämfört med barn med en negativ födoämnesprovokation. Såväl före som efter provokationsserien kunde högre nivåer av inflammationsmarkörerna eosinofil-deriverat neurotoxin (EDN) och kalprotektin uppmätas i avföringsprover från barn med positivt provokationsutfall jämfört med barn med negativ födoämnesprovokation. Skillnaderna i uppmätta nivåer av biomarkörer i faeces uppnådde dock inte statistisk signifikans. Slutsats: Rapporterad födoämnesöverkänslighet var vanligt förekommande bland skolbarn i Norrbotten och ökade från 7-8 år till 11-12 års ålder. Incidensen av rapporterad födoämnesöverkänslighet var hög, liksom remissionen. Prevalensen av rapporterad allergi mot mjölk, ägg, fisk eller vete var 8 gånger högre än den prevalens som kunde påvisas med dubbelblind placebokontrollerad födoämnesprovokation. Allergi mot mjölk, ägg, fisk och vete var en ovanlig orsak till att barn helt undvek dessa födoämnen på grund av upplevd överkänslighet. Några av de biomarkörer som analyserades innan provokationsserierna visade lovande resultat som möjliga, framtida prognostiska markörer för symptomatisk, IgE-medierad födoämnesallergi. Dessa resultat behöver dock valideras med ytterligare studier.

Cachexia affects up to two thirds of all cancer patients with progressive disease. It is a syndrome characterised by weight-loss, anorexia, fatigue, asthenia, peripheral oedema, and is responsible for around 20% of cancer deaths. Cachectic patients suffer loss of both muscle mass and adipose tissue (with comparative sparing of visceral protein) and the lean tissue loss appears resistant to nutritional support. Progress in the treatment of cancer cachexia has been hampered due to poor understanding of the molecular mechanisms of skeletal muscle wasting in humans (rather than preclinical models) combined with a lack of accurate phenotyping particularly with respect to loss of skeletal muscle mass and function. The aim of the present thesis was to improve the knowledge and tools available for early intervention studies. The thesis focused on skeletal muscle as a key compartment in cancer cachexia. The experimental model was patients with upper gastrointestinal (UGI) cancer undergoing potentially curative surgery due to the associated higher incidence of cachexia along with the ability to access tissue biopsies. The thesis broadly divides into two sections. Part I reports a series of cancer cachexia biomarker discovery studies based on direct biopsy and analysis of human skeletal muscle. Part II focused on assessment and phenotyping of skeletal muscle mass and function in cachectic UGI cancer patients. In addition, the feasibility of longitudinal clinical studies that utilise such methodology is reported. Intramuscular β-dystroglycan protein content (assessed using Western blot) was identified as a potential biomarker of cancer cachexia whereas changes in the structural elements of muscle (myosin heavy chain or dystrophin) appeared to be survival biomarkers. Using transcriptomics, an 82-gene signature was demonstrated to correlate with weight-loss. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to examine the genes from this signature that were most upregulated. The exercise activated genes, CAMk2β and TIE1, correlated positively with weight-loss across different muscle groups (Rectus abdominis, Vastus lateralis, Diaphragma) indicating that cachexia was not simply due to inactivity and suggesting that these genes could be used as biomarkers of cachexia. None of the biomarkers discovered were consistent with pre-clinical models and therefore require further study before progressing to a validation programme. Electron microscopy of muscle biopsies demonstrated that the number and size of intramyocellular lipid droplets was increased in the presence of cancer and increases further with weightloss/ loss of adipose mass in other body compartments. The specific mechanisms and drivers of this phenomenon remain to be elucidated, but could relate to enhanced lipolysis or mitochondrial dysfunction in skeletal muscle as well as influencing muscle mechanical quality. Physiological assessment of patients with cancer cachexia established the negative impact that cachexia can have on muscle mass, function, muscle quality and quality of life, but demonstrated that the degree of impairment varies with sex and between muscle groups. Furthermore, the challenge of longitudinal studies in this patient group where frailty and clinical deterioration limit repeated assessments was highlighted. Such issues emphasise the need for a dual approach to the classification of cancer cachexia: if molecular markers prove difficult to discover or validate, then more specific and robust physiological indices of skeletal muscle mass and function may be the more important route to improve clinical trial design and cachexia classification.

Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-30 22:46:35.072

Many blood markers have been associated with stroke. I set out to determine whether blood markers can be applied to: (i) improve the accuracy of the clinical diagnosis of stroke or TIA, and/or (ii) improve the prediction of poor outcome in patients who are still symptomatic at the time of admission with stroke or TIA. I systematically reviewed the existing literature on the diagnostic performance of a range of blood markers measured soon after stroke onset, to inform the choice of markers for my subsequent prospective studies in this thesis. Many studies had deficiencies in their design, which may have explained the apparently – and perhaps spuriously - impressive diagnostic performance of several markers. In the light of these data I was able to improve the design of my own studies and suggest how future studies of diagnostic markers could be improved. In order to define an appropriate comparator test for assessing the diagnostic accuracy of blood markers, I first examined the performance of emergency room nurses and doctors. I assessed the accuracy of their diagnosis of TIA or stroke (‘acute cerebrovascular disease’) in patients presenting with symptoms of suspected stroke, and compared them with a number of stroke diagnostic scales. In the 405 patients recruited to the study, the sensitivity of emergency department staff was 77% and specificity 58%. Each stroke diagnostic scale had a slightly better sensitivity, though worse specificity, than an emergency department clinician. I decided to use the diagnosis by an emergency department clinician of ‘probable or definite acute cerebrovascular disease’ as the best clinical performance reference standard. In blood taken from the same cohort of 405 patients, accredited research laboratories measured markers of inflammation, thrombosis, thrombolysis, cardiac strain and cerebral damage. Tissue plasminogen activator and loge N-terminal pro brain natriuretic peptide were associated positively with a diagnosis of acute cerebrovascular disease, though each marker did not add diagnostic value to the diagnosis of an emergency department doctor or nurse. I systematically reviewed the literature examining the association between the levels of blood markers with poor outcome (i.e. death or dependency) after stroke. I found that although almost all markers studied had a positive association with poor outcome, there were methodological problems with many studies, chiefly small sample size, publication bias or within study reporting biases, and lack of adjustment for important confounders such as age or stroke severity. With data from the Edinburgh Stroke Study, I examined the association between circulating markers of the inflammatory response (white cell count, interleukin-6, Creactive protein and fibrinogen) and poor outcome after stroke. After adjustment for age, whether the patient lived alone, was independent of activities of daily living, was orientated, able to lift both arms and able to walk, I found that higher levels of interleukin-6, white cell count and glucose were associated with poor outcome. The relevant test of a biological marker is not its predictive ability alone, but whether, when added to a validated predictive model based on clinical variables, it improves the prediction of outcome. No individual marker improved the prediction of poor outcome when added to a validated prognostic model based on clinical variables alone. From my cohort of 405 patients with suspected stroke 285 patients had a confirmed diagnosis. Follow up of these 285 patients with confirmed acute cerebrovascular disease showed that, after adjustment for neurological impairment and age, only interleukin-6 and N-terminal pro brain natriuretic peptide were significantly associated with death or disability at 3 months. Neither marker improved the predictions of a model to predict poor outcome based on clinical variables alone. To examine the relationship between circulating markers of the inflammatory response and recurrent stroke, myocardial infarction, and vascular death (‘recurrent vascular events’), again I used data from the Edinburgh Stroke Study. After adjustment for clinical predictors (age, prior MI, stroke, or TIA and AF) I found that higher levels of interleukin-6, C-reactive protein and fibrinogen remained significantly associated with an increased risk of recurrent vascular events. However, the relationship with deaths from all causes was somewhat stronger for each marker, perhaps suggesting that higher marker levels were associated with debility rather than vascular events per se. In conclusion, I found no marker measured could improve on the diagnostic accuracy of an emergency department clinician for acute cerebrovascular disease, nor improve the prediction of poor outcome by a prognostic model based upon clinical variables. The work of this thesis does not support the routine use of blood markers as an aid to the diagnosis of, or the prediction of outcome of, acute stroke.

Background: The study of circulating biomarkers and their association with disease outcomes has become progressively complex due to advances in the measurement of these biomarkers through multiplex technologies. The Least Absolute Shrinkage and Selection Operator (LASSO) is a data analysis method that may be utilized for biomarker selection in these high dimensional data. However, it is unclear which LASSO-type method is preferable when considering data scenarios that may be present in serum biomarker research, such as high correlation between biomarkers, weak associations with the outcome, and sparse number of true signals. The goal of this study was to compare the LASSO to five LASSO-type methods given these scenarios. Methods: A simulation study was performed to compare the LASSO, Adaptive LASSO, Elastic Net, Iterated LASSO, Bootstrap-Enhanced LASSO, and Weighted Fusion for the binary logistic regression model. The simulation study was designed to reflect the data structure of the population-based Tucson Epidemiological Study of Airway Obstructive Disease (TESAOD), specifically the sample size (N = 1000 for total population, 500 for sub-analyses), correlation of biomarkers (0.20, 0.50, 0.80), prevalence of overweight (40%) and obese (12%) outcomes, and the association of outcomes with standardized serum biomarker concentrations (log-odds ratio = 0.05-1.75). Each LASSO-type method was then applied to the TESAOD data of 306 overweight, 66 obese, and 463 normal-weight subjects with a panel of 86 serum biomarkers. Results: Based on the simulation study, no method had an overall superior performance. The Weighted Fusion correctly identified more true signals, but incorrectly included more noise variables. The LASSO and Elastic Net correctly identified many true signals and excluded more noise variables. In the application study, biomarkers of overweight and obesity selected by all methods were Adiponectin, Apolipoprotein H, Calcitonin, CD14, Complement 3, C-reactive protein, Ferritin, Growth Hormone, Immunoglobulin M, Interleukin-18, Leptin, Monocyte Chemotactic Protein-1, Myoglobin, Sex Hormone Binding Globulin, Surfactant Protein D, and YKL-40. Conclusions: For the data scenarios examined, choice of optimal LASSO-type method was data structure dependent and should be guided by the research objective. The LASSO-type methods identified biomarkers that have known associations with obesity and obesity related conditions.

This thesis was focused on testing of portable Raman spectrometer for detection of components in model mixtures of five different biomolecules in a gypsum matrix. The main goal was to test the ability of the instrument to detect amino acids glycine, L-proline, L-alanine and nucleobase thymine in samples containing UV protective pigment -carotene. Other measurements were taken using the laboratory inVia Reflex instrument to comapare the results gained from the portable Raman spectrometer. Both instruments were equipped with the excitation laser working at 785 nm wavelength. It was found out that it is possible to detect amino acid in mixtures containing -carotene at the lowest concentration of amino acid 100 g/kg and the maximum concentration of -carotene 1 g/kg. Thymine could be identified at the concentration of 100 g/kg in mixtures with higher concentration of -carotene (10 g/kg). In mixtures of -carotene at the concentration of 1 g/kg and only one of the studied nitrogen containg compounds (at the concentration of 100 g/kg) in the gypsum matrix, it was possible to detect all present components. In mixtures containing two nitrogen compounds with -carotene at the concentration of 1 g/kg in the gypsum matrix, both nitrogenous compounds could be detected. When measuring the samples of the three nitrogen...

The study was initiated to examine magnesium intake, supplementation and their relation to inflammation. Magnesium is the second most abundant extracellular ion following potassium. Outside the cell, magnesium can be found in bone tissue, cardiac muscle tissue, other tissues and in the blood. Magnesium form compounds which operate in several essential metabolic processes in the body. Magnesium deficiency may have an impact on insulin resistance and endothelial dysfunction, which may result in an increased level of inflammation. Increased inflammation over a longer period has been seen to increase the risk of common lifestyle induced diseases such as diabetes type II and coronary heart diseases. The study of magnesium and its influence on inflammation is thereby becoming important and interesting for all societies and in their effort to find solutions to maintain and increase the well-being of its individuals. The study is a literature study based on searches made in One Search and Pub Med databases. A total of ten studies were included, five for magnesium intake and five for supplementation. The majority of the studies showed a significant correlation between increased magnesium intake, dietary and supplementary, with decreased levels of inflammatory biomarkers and hints that magnesium might have a role in the inflammation process. What needs to be taken into account is that fiber intake in two studies attenuated magnesium’s inverse relation to inflammation. In addition of a decrease in inflammatory biomarker levels the risk for developing diabetes type II seemed to decrease as well with an increased intake of magnesium in one of the studies. Further studies need to be executed in order to establish the role of magnesium in inflammation and optimal dosage for prevention of metabolic and cardiovascular diseases. / Studien undersöker magnesiumintag och supplementering med magnesium samt dess inverkan på inflammation. Magnesium är den vanligast förekommande jonen intracellulärt efter kalium.  Extracellulärt magnesium förekommer i benvävnad, hjärtmuskelvävnad och i blodet. Magnesium bildar ämnen som medverkar i flera viktiga metabola processer i kroppen. Magnesiumbrist kan ha en inverkan på insulin resistans och endotel dysfunktion som följaktligen skulle kunna resultera i en ökad nivå av inflammation. Ökad inflammation under en längre tid har visat sig öka risken för vanliga livsstilssjukdomar som diabetes typ II och hjärt- och kärlsjukdomar. Forskning om magnesium och dess effekt på inflammation blir därmed viktig och intressant för samhällen i deras strävan att hitta lösningar till att bibehålla och öka välmåendet hos populationen. Studien är en litteraturstudie och är grundad på sökningar via databaserna One Search och Pub Med. Totalt tio studier inkluderades i arbetet, fem som undersökte magnesiumintag och inflammation samt fem som undersökte supplementering av magnesium och inflammation. Majoriteten av studierna visade på en signifikant korrelation mellan ett ökat magnesiumintag, via kosten och kosttillskott, och minskade nivåer av biomarkörer för inflammation. Det antyder att magnesium kan ha en roll i inflammationsprocessen. I de två studier som mätte fiberintaget var relationen mellan magnesiumintag och inflammation försvagad. Utöver en minskning av biomarkörer för inflammation sågs en minskad risk för att utveckla diabetes typ II vid ett ökat magnesiumintag i en av studierna. Fler studier krävs för att fastställa magnesiums betydelse vid inflammation samt den optimala doseringen för prevention av metabola och kardiovaskulära sjukdomar.