Die systemische Verabreichung von Amoxizillin/Metronidazol (AM) als Zusatz zu Scaling/Root planing (SRP) in der Behandlung der generalisierten Aggressiven Parodontitis (gAP) führt zu guten klinischen und mikrobiologischen Ergebnissen. Die lokale antiseptische Zusatzmedikation mit Chlorhexidin in einem Controlled-Delivery-Device (PerioChip, PC) verbessert das Ergebnis von SRP in der Behandlung der Chronischen Parodontitis. Die Anwendung in der Behandlung der gAP ist bisher noch nicht untersucht worden. Ziele: Der Effekt von PC als Zusatz zu SRP in der Therapie der gAP wurde untersucht. Die Wirksamkeit wurde mit der Standardmedikation AM verglichen. Neben klinischen Parametern wurde die Konzentration des Entzündungsmarkers Calprotectin in der Sulkusflüssigkeit (SF) bestimmt. Material/Methode: 36 gAP-Patienten (18/Gruppe, 35+/-4 Jahre) wurden mit SRP und randomisiert entweder mit AM oder PC behandelt. Zur Baseline, 3 und 6 Monate nach SRP wurden die klinischen Parameter PD, CAL, BoP, Pus erhoben sowie SF-Proben tiefer und flacher Referenzstellen entnommen. Die Calprotectin-Konzentration in der SF wurde mittels ELISA bestimmt. Ergebnisse: 3 Monate nach SRP zeigten beide Gruppen signifikante Verbesserungen der klinischen Parameter, wobei noch keine Unterschiede zwischen den Gruppen bestanden. Während die AM-Gruppe stabil blieb, verschlechterten sich mehrwurzelige Zähne und tiefe Referenzstellen der PC-Gruppe wieder signifikant. Nach 6 Monaten wies die AM-Gruppe signifikant mehr CAL-Gewinn und PD-Reduktion auf. Pus war nur noch in der PC-Gruppe nachweisbar. Die Calprotectin-Konzentration war zwar in beiden Gruppen signifikant gesunken, Unterschiede zwischen den Gruppen bestanden jedoch aufgrund hoher Standardabweichungen nicht. Schlussfolgerung: AM zeigte eine bessere klinische Wirksamkeit und Langzeitstabilität als PC. Aufgrund großer interindividueller Variabilität spiegelte die SF-Konzentration von Calprotectin die klinischen Unterschiede nicht wider. / Adjunctive systemic administration of amoxicillin/metronidazole (AM) in generalized aggressive periodontitis (gAP) therapy results in good clinical and microbiological outcome. Adjunctive use of chlorhexidine within a controlled-delivery-device (PerioChip, PC) improves the outcome of scaling/root planing (SRP) in chronic periodontitis therapy. Its effect in the treatment of gAP has not been evaluated. Aims: The effect of adjunctive use of PC in the treatment of gAP was investigated. Efficacy of PC was compared to the standard treatment with AM. Clinical parameters and the concentration of the inflammation marker calprotectin within gingival crevice fluid (GCF) were measured. Material/methods: 36 gAP patients (18/group, 35+/-4 years) were treated by SRP either AM or PC. Clinical parameters PD, CAL, BoP and Pus were recorded at baseline, 3 and 6 months after therapy. GCF was sampled at deep and shallow reference sites and the concentration of calprotectin was measured by ELISA. Results: Both groups showed significant improvements of clinical parameters 3 months after SRP, however differences between groups were not significant. While the AM-group remained clinically stable, multirooted teeth and deep reference sites of the PC-group showed significant deterioration again. 6 months after SRP, the AM-group showed significant more CAL gain and PD reduction. Pus was detectable in the PC group only. The concentration of calprotectin in GCF had significantly decreased in both groups; however differences between groups were not significant due to high standard deviations. Conclusion: AM showed higher efficacy in terms of clinical treatment outcome and long-term stability than PC. Due to high interindividual variability, GCF-concentrations of calprotectin failed to reflect clinical differences.

The concentration of calprotectin in feces (fCal) is a clinically useful marker of chronic gastrointestinal inflammation in humans and dogs. No commercial assay is widely available to measure fCal in small animal medicine, to date. Thus, we verified the immunoturbidimetric fCAL turbo assay (Bühlmann) of fCal for canine and feline fecal extracts by determining linearity, spiking and recovery, and intra-assay and inter-assay variability. We determined RIs, temporal variation over 3 mo, and effect of vaccination and NSAID treatment. Observed:expected (O:E) ratios (x̄ ± SD) for serial dilutions of feces were 89– 131% (106 ± 9%) in dogs and 77–122% (100 ± 12%) in cats. For spiking and recovery, the O:E ratios were 90–118% (102 ± 11%) in dogs and 83–235% (129 ± 42%) in cats. Intra- and inter-assay CVs for canine samples were ≤19% and ≤7%, and for feline samples ≤22% and ≤21%. Single-sample RIs were <41 μg/g for dogs and <64 μg/g for cats. With low reciprocal individuality indices, using population-based fCal RIs is appropriate, and moderate fCal changes between measurements (dogs 44.0%; cats: 43.2%) are considered relevant. Cats had significant (but unlikely relevant) fCal increases post-vaccination. Despite individual fCal spikes, no differences were seen during NSAID treatment. The fCAL turbidimetric assay is linear, precise, reproducible, and sufficiently accurate for measuring fCal in dogs and cats. Careful interpretation of fCal concentrations is warranted in both species during the peri-vaccination period and for some patients receiving NSAID treatment.

Background: Colorectal cancer (CRC) is the third most common cancer in men and the second most common in women worldwide. Adenomas can be precursors to CRC, and inflammatory bowel disease (IBD) can present with the same symptoms as CRC. The majority of patients with CRC initially consult primary care. Symptoms associated with CRC are also common among primary care patients, but seldom caused by any significant disease. Reliable diagnostic aids would be helpful in deciding which patients to refer. Faecal immunochemical tests (FITs) are commonly used for this purpose in primary care in Sweden, but there is little evidence to support this use. Faecal calprotectin (FC) has been suggested as an additional test. Aim: To explore how doctors in primary care investigate patients with suspected CRC, the value of FITs, symptoms and presence of anaemia in diagnosing CRC and adenomas in primary care, and whether FC tests could contribute to diagnosis. Methods: Three studies (1-3) were carried out in Region Jämtland Härjedalen, Sweden. There was no screening programme for CRC. We used a point of care qualitative dip-stick 3-sample FIT with a cut-off of 25-50μg haemoglobin/g faeces, and a calprotectin enzyme-linked immunosorbent assay (ELISA) test with a cut-off of 100 μg/g faeces. 1: A retrospective, population-based study including all patients diagnosed with CRC or adenomas with high-grade dysplasia (HGD) during the period 2005-2009 that initially consulted primary care. Symptoms, FIT results, anaemia and time to diagnosis were retrieved from medical records. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated from FIT results at the region’s health centres 2008- 2009. (Paper I.) 2: A prospective cohort study including consecutive patients where primary care doctors requested FITs and/or FC tests, at four health centres, from 30 Jan 2013 to 31 May 2014. FITs, FC tests, haemoglobin and iron deficiency tests were analysed; patients and doctors answered questionnaires about symptoms. Patients were examined with bowel imaging or followed for two years. Findings of CRC, adenomas with HGD, adenomas with low grade dysplasia (LGD) ≥1 cm and IBD were registered. (Papers II and III.) 3: A qualitative study of interviews with eleven primary care doctors. We explored what made them suspect CRC, and their practices regarding investigation and referral with particular attention to their use of FITs. Qualitative content analysis with an inductive approach was used for the analysis. (Paper IV.) Results: 1: Paper I: Of 495 patients 323 (65.3%) started the investigation in primary care. FITs were analysed in 215. In 23 cases with CRC, FITs were negative; 15 (65.2%) had anaemia. In 33 cases with CRC, FITs were performed due to asymptomatic anaemia; 10 (30.3%) had negative FITs. The time from start of investigation, to the diagnosis of CRC or adenomas with HGD, was significantly longer for patients with negative FITs. 2: 377 patients (9 diagnosed with CRC, 10 with IBD) were included. Paper II: Concordance of positive answers about symptoms from patients and doctors was generally low. Rectal bleeding (recorded by 43.5% of patients and 25.6% of doctors) was the only symptom related to CRC and IBD. The FIT showed a better PPV than rectal bleeding for CRC and IBD. When patients recorded rectal bleeding, the FIT had a PPV of 22.6% and a NPV of 98.9% for CRC and IBD. Paper III: The best test for detecting CRC and IBD was the combination of a positive FIT and/or anaemia with a sensitivity, specificity, PPV and NPV of 100%, 61.7%, 11.7% and 100% respectively. The FC test had no additional value to the FIT alone. The sensitivity, specificity, PPV and NPV of the FIT for CRC in study 1 was estimated at 88.4%, 73.3%, 6.2% and 99.7% respectively. In study 2, corresponding figures were 88.9%, 67.4%, 6.3% and 99.6% respectively. 3: Paper IV: We identified four categories: “Careful listening – with awareness of the pit-falls”, “tests can help – the FIT can also complicate the diagnosis”, “to refer or not to refer – safety margins are necessary”, and “growing more confident – but also more humble”. All doctors had found their own way to handle FIT results in the absence of guidelines. Conclusion: The diagnostic process when suspecting CRC can be described as navigating uncertain waters with safety margins. FITs were often used by primary care doctors but with considerable variations in interpretation and handling of results. Rectal bleeding was the only symptom related to CRC and IBD, but the FIT showed a better PPV than rectal bleeding. The combination of a negative FIT and no anaemia may be useful as a rule-out test when CRC is suspected in primary care, and this potentially also applies when patients present with rectal bleeding. Further studies are needed to confirm this and to determine the optimal FIT cut-off value for this use.

This thesis describes studies in patients with inflammatory bowel disease (IBD) and collagenous colitis (CC). We investigated mucosal eosinophil and neutrophil granulocytes and T-cells involved in the inflammatory processes and aimed at determining whether these processes are reflected in the faecal (F) contents of specific proteins secreted by cells in the intestinal mucosa. Thus, we measured eosinophil cationic protein (ECP) and eosinophil protein X (EPX) and the neutrophil derived myeloperoxidase (MPO) and calprotectin (C); and in addition, chromogranin A (CgA), Chromogranin B (CgB) and secretoneurin (SN), derived from EEC cells and cells in the enteric nervous system. We found that a normalised FC level can serve as a surrogate marker for successful treatment in patients with IBD, but persistently high FC levels need further evaluation (study I). Furthermore, FC and F-MPO appear to relate better than F-EPX to treatment outcome in IBD. We evaluated F-ECP, F-EPX, F-MPO and FC as markers of disease activity and treatment outcome in patients with CC (study III) and concluded that F-ECP was the best discriminator of detecting active CC. Normalised F-ECP and F-EPX could serve as markers of successful treatment. We showed that the inflammation in CC is characterised by activated eosinophils, but that there is no neutrophil activity (study II). T-cells have a lower grade of activity in active CC than in control subjects. During budesonide treatment the normal activation of eosinophils and T-cells is restored, with concomitant clinical remission. The findings in studies II and III indicate that the eosinophils have an essential role in the pathophysiology of CC. Markedly higher values of F-CgA, F-CgB and F-SN were found in patients with CC than in those with IBD and controls (study IV) indicating a crucial role for the intestinal neuro-endocrine system in the pathogenesis of collagenous colitis.

Procyanidins (PCs) have been extensively investigated for their potential health protective activities, but the prospective bioactivities are limited by their poor bioavailability. The majority of the ingested dose remains unabsorbed and reaches the colon where extensive microbial metabolism occurs. The objectives of these studies are to better understand the roles and activities of PCs in the lower gastrointestinal tract. First, a new high-throughput Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry method was developed to efficiently analyze PCs and an extensive profile of their microbial metabolites. This method is sufficiently sensitive and effective in simultaneously extracting and measuring native PCs and their microbial metabolites in biological samples. Furthermore, administration of grape seed extract increased the expression of gut junction protein occludin and reduced levels of fecal calprotectin, which suggests an improvement of gut barrier integrity and a potential modulation of endotoxemia. Additionally, chronic supplementation of the diet with flavanols did not increase colonic tissue accumulation of PCs or their microbial metabolites over a 12 week feeding study. This was the first long-term study of its kind, and the results indicate that we still do not fully understand the outcome of ingested flavanols in the colon during chronic exposure rather than acute doses. Lastly, new understanding of the microbial metabolism of PCs in the colon has been reached by studying the colon as 4 segments, rather than as a complete unit as previous studies have done. Data show that a gradient is established along the length of the colon for both PCs and their metabolites, with PCs reaching highest concentrations within 3 h after ingestion, while metabolites reach maximum concentrations anywhere form 3-18 h after ingestion. Moreover, data indicate the progressive, step-wise degradation of PCs into small metabolites throughout the length of the colon. Overall, there is greater understanding of the colonic metabolism of dietary PCs derived from GSE and cocoa, the accumulation of these compounds, and their effect on gut permeability. Future work will build off of these novel studies, and will continue to advance the understanding of the health benefits of dietary PCs. / Ph. D.

Laboratory analyses, essential in screening, diagnosis, treatment, and monitoring of disease, are indispensable in health care, but appropriate utilization is intricate. The overall aim of this thesis was to study the use of laboratory tests in Sweden with the objective to evaluate and optimize test utilization. Considerable inter-county variations in test utilization in primary health care in Sweden were found; variations likely influenced by local traditions and habits of test ordering leading to over- as well as underutilization. Optimized test utilization was demonstrated to convey improved quality and substantial cost savings. It was further established that continuing medical education is a suitable means of optimizing test utilization, and consequently enhancing quality and cost-efficiency, as such education was demonstrated to achieve long-lasting improvements in the test ordering habits of primary health care physicians. Laboratory tests are closely associated with other, greater, health care costs, but their indirect effects on other areas of medicine are rarely evaluated or measured in monetary terms. In an illustrative example of the effects that optimal test utilization may have on associated health care costs it was demonstrated that F-calprotectin, a fecal marker of intestinal inflammation, has the potential to substantially reduce the number of invasive investigations necessary in, and the costs associated with, the diagnosis of Inflammatory Bowel Disease. Information on trends in test utilization is essential to optimal financial management of laboratories. A longitudinal evaluation revealed that test utilization had increased by 70% in 6 years, and even though the selection of tests more than doubled, a very small number of tests represented a stable, and disproportionally large, share of the total number of tests ordered. The study defines trends and thus has potential predictive values. In summary, appropriate utilization of laboratory analyses has both clinical and economical benefits on all levels of health care.

Les neutrophiles peuvent synthétiser et relâcher l’angiopoïétine 1 (Ang1), un facteur de croissance cytosolique impliqué dans l’angiogenèse, capable d’induire plusieurs activités pro-inflammatoires chez les neutrophiles. Ces derniers induisent la synthèse et la relâche de neutrophil extracellular traps (NETs), des filaments d’ADN nucléaires décondensés transportant des protéines telles que l’élastase, la myélopéroxydase (MPO), la protéinase (PR3) et la calprotectine (S100A8/S100A9) qui contribuent à la réponse immunitaire innée afin de combattre les pathogènes (i.e. bactéries). Les NETs ont des effets pro-inflammatoires, pro-thrombotiques, jouant un rôle dans la dysfonction endothéliale, et ont récemment été retrouvés dans l’insuffisance cardiaque (IC) et le diabète de type 2 (T2DM). Le but de cette étude est de déterminer la synthèse et la relâche d’Ang1 chez des patients T2DM et insuffisants cardiaques avec fraction d’éjection préservée (HFpEF) (stables ou en décompensation aigue (ADHFpEF)) avec ou sans T2DM en comparant à des volontaires sains (VS) comme groupe témoin. Nos résultats démontrent qu’en absence de traitement (PBS) et avec un traitement avec LPS, les niveaux de NETs augmentent chez les patients ADHFpEF + T2DM comparé aux VS. Nous observons également que le LPS, le PMA ou A23187 augmentent la synthèse de l’Ang1 (de 150 à 250%) chez des VS et cet effet est amplifié chez les patients T2DM et IC. L’Ang1 est relâchée à 100% par les neutrophiles de tous les groupes à l’étude et ne se lient pas sur les NETs, comparé à la calprotectine. Notre étude suggère que les patients en insuffisance cardiaque aigue (ADHFpEF + T2DM) synthétisent et relâchent plus de NETs et que l’exocytose de l’Ang1 est indépendante de la synthèse et la relâche de NETs. / Neutrophils can induce the synthesis and release of angiopoietin 1 (Ang1), a cytosolic growth factor involved in angiogenesis capable of inducing several neutrophil-driven pro-inflammatory activities. Neutrophils also synthesize and release neutrophil extracellular traps (NETs) composed of decondensed nuclear DNA filaments carrying proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), proteinase 3 (PR3) and calprotectin (S100A8/S100A9) which contribute to the innate immune response to combat pathogens (e.g., bacteria). NETs are also implicated in various pathological conditions through pro-inflammatory, pro-thrombotic properties, leading to endothelial dysfunction, and have recently been found in heart failure (HF) and type 2 diabetes (T2DM). The purpose of this study is to determine the synthesis and release of Ang1 in patients with T2DM and HF with preserved ejection fraction (HFpEF) (stable or acute decompensated (ADHFpEF)) with or without T2DM, compared to healthy volunteers (HV) as control group. Our results show that in absence of agonist (PBS) and after LPS treatment, NETs levels are increased in ADHFpEF + T2DM patients compared to HV. We also observed that LPS, PMA or A23187 treatments increase the synthesis of Ang1 (from 150 to 250%) in HV and this effect is amplified in T2DM and HF patients. Ang1 is completely released (100%) by neutrophils isolated from all cohorts and does not bind to NETs compared to calprotectin. Our study suggests that severely ill patients with decompensated heart failure (ADHFpEF + T2DM) synthesize and release more NETs and that Ang1 exocytosis is independent from NETs synthesis.

Kalprotektin är ett protein som bygger upp 60 % av neutrofila granulocyters granula. Vid inflammationer i tarmen vandrar neutrofila granulocyter ut genom mukosan och följer med faeces ut ur kroppen. Proteinet är kalciumbindande och surt och dessa egenskaper utnyttjas i moderna extraktionskit för att mäta mängden kalprotektin i faeces (F-kalprotektin). Thermofisher är ett företag som utvecklat en metod för analys av F-kalprotektin och den kallas för EliA Extraction kit 2 där Kalprotektin extraheras med hjälp av bufferten i röret och analyseras därefter av Phadia 250. Syftet med studien var att utföra en metodjämförelse mellan två metoder. I den första metoden kommer den rutinmetod som används på laboratoriet användas för att upparbeta faeces. I den andra kommer patienterna som skickar sin faeces direkt samlade i extraktionsrören till analys. Syftet är att jämföra analysresultaten för att se om de är lika. Studien utfördes på 26 vuxna patienter varav 11 var kvinnor och 15 var män. Innan analys av prover analyserades tre kontroller för att kontrollera om metoden för analys av F-kalprotektin fick analyseras. Under studien utfördes fyra olika inom-serie precision på fyra olika patienter med 25 replikat för att beräkna variationskoefficienten (CV). Metodens CV varierade mellan 19 - 27 %. Efter att en reparation på instrumentet utförts analyserades två prover med inom-serie precision och CV blev 8 – 13 %. Metodernas resultat hade stark korrelation (R = 0,9055) och vid beräkning av resultat med Mann-Whitney U-test sågs ingen signifikant skillnad (p = 0,3059). Det fanns dock en del prover som hade stor skillnad på resultatet vilket kan ge fel diagnos/klassning. För att kunna bedöma om den nya metoden kan användas som ett alternativ till rutinmetoden måste en större population analyseras. / Calprotectin is a protein that makes up 60 % of the granules of neutrophilic granulocytes. In inflammation of the intestine, neutrophilic granulocytes migrate out through the mucosa and follow the feces out of the body. The protein is calcium-binding and acidic and these properties are used in modern extraction kits to measure the amount of calprotectin in feces  (F-calprotectin). Thermofisher is a company that has developed a method for analysis of F-calprotectin and it is called EliA Extraction kit 2 which is analyzed by Phadia 250. The purpose of the study was to perform a method comparison between two methods. In the first method, the routine method used in the laboratory will be used to process feces. In the second, the patients who send their feces directly collected in the extraction tubes come for analysis. The purpose is to compare the analysis results to see if they are similar. The study was performed on 26 individuals of which 11 were women and 15 were men. Before analyzing, three controls were analyzed to check the method of analysis of F-calprotectin. A negative check and a positive check as well as a calibration check. During the study, four different within-run precision were performed on four different patients with 25 replicates to calculate the coefficient of variation (CV). The CV of the method varied between 19 – 27 %. After a repair on the instrument was performed, the CV was 8 – 13 %. The results of the methods had a strong correlation (R = 0.9055) and when calculating the results with Mann-Whitney U-test, no significant difference was seen (p = 0.3059). In order to be able to assess whether the new method can be used as an alternative to the routine method, a larger population must be analyzed.