Biomarkers in endotoxemia with a special interest in citrullinated histone H3

Background: Sepsis with multi-organ failure has an unacceptably high mortality rate of 25- 30% despite the use of antibiotics and modern intensive care. Attenuating the multi-organ failure and finding new biomarkers for early detection of septic shock would be of high clinical relevance in order to improve outcome. Aim: The aim of this thesis was to study if inhaled nitric oxide (iNO) in combination with steroids could attenuate multi-organ failure in a porcine model of endotoxemia. We further aimed to investigate the dynamics of circulating citrullinated histone H3 (H3Cit), a recently proposed biomarker in sepsis, in a human model of endotoxemia. Since microvesicles (MVs) have been shown to be elevated in sepsis, a further objective of this thesis was to investigate whether H3Cit could be detected bound to MVs. Methods: A randomized controlled trial (RCT) with 30 domestic piglets exposed to lipopolysaccharide (LPS)-alone, LPS + iNO, LPS + IV steroid, LPS + iNO + IV steroid or anesthesia only (Control) was conducted in paper I. Various biomarkers were measured at endpoint in order to evaluate organ function after 30 hrs of endotoxic shock. In paper II, an ELISA-based assay quantifying plasma H3Cit was developed and methodologically validated in accordance to recommended ELISA validation requirements. This ELISA, as well as a flow cytometric assay quantifying MV-bound H3Cit, was used in plasma samples at baseline and then 2, 4 and 7 hrs after LPS-injection in a placebo controlled RCT including 22 healthy volunteers in Paper III. Results: LPS + iNO+ IV steroid tended to require less norepinephrine and were significantly less acidotic (p < 0.05) compared to LPS-only in the porcine model of endotoxemia. No significant differences could, however, be detected in other clinical variables. Circulating H3Cit, quantified by the ELISA assay, which was validated with high specificity, precision and stability, rose significantly after LPS injection in the human model of endotoxemia. Similar elevations were seen when quantifying H3Cit-bearing neutrophil- and platelet derived MVs. Conclusions: Our data suggest that combined therapy with iNO and IV steroid is at least partially, protective after experimental LPS infusion. We furthermore show that circulating H3Cit, quantified by two distinct methods, is elevated after experimental LPS injection, suggesting that H3Cit may be a novel biomarker even in sepsis. Our data also show that H3Cit can be detected bound to MVs, proposing a novel mechanism by which H3Cit can be transported throughout the vasculature

Nine-Tenths of the Law: The English Copyright Debates and the Rhetoric of the Public Domain

Background: The role of vitamin D supplementation as adjuvant treatment of tuberculosis (TB) has lately attracted increasing interest. Our aim was to investigate the capacity of alveolar macrophages (AMs) from patients with or without exposure to TB to control intracellular growth of virulent Mycobacterium tuberculosis (Mtb). Methods: AMs were freshly harvested from the bronchoalveolar lavage fluid of 7 patients with a history of TB (4 patients with previous TB and 3 patients with current TB) and 4 non-TB subjects. The H37Rv strain, genetically modified to express Vibrio harveyi luciferase, was used to determine the growth of Mtb by luminometry in the AMs from study subjects. Cytokine levels in culture supernatants were determined using a flow cytometry-based bead array technique. Results: AMs from patients with a TB history were less efficient in restricting Mtb growth. Stimulation with 100 nM1, 25-dihydroxyvitamin D (1,25D3) did not significantly influence the capacity of AMs from any study subjects to control the infection. Out of the cytokines evaluated (TNF-α, IL-1β, IL-10 and IL-12p40) only TNF-α demonstrated detectable levels in culture supernatants, but did not respond to stimulation with 1,25D3. Conclusions: We conclude that AMs of TB-patients show reduced ability to control mycobacterial growth in vitro, and, that AMs in this pilot study do no respond to 1, 25D3-stimulation. The former observation supports the concept that innate immunity is crucial for the control of TB infection

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OBJECTIVE: It has previously been shown that a combination of inhaled nitric oxide (iNO) and intravenous (IV) steroid attenuates endotoxin-induced organ damage in a 6-hour porcine endotoxemia model. We aimed to further explore these effects in a 30-hour model with attention to clinically important variables. DESIGN: Randomized controlled trial. SETTING: University animal laboratory. SUBJECTS: Domestic piglets (n = 30). INTERVENTIONS: Animals were randomized into 5 groups (n = 6 each): 1) Controls, 2) LPS-only (endotoxin/lipopolysaccharide (LPS) infusion), 3) LPS + iNO, 4) LPS + IV steroid, 5) LPS + iNO + IV steroid. MEASUREMENTS AND MAIN RESULTS: Exposure to LPS temporarily increased pulmonary artery mean pressure and impeded renal function with elevated serum creatinine and acidosis compared to a control group over the 30-hour study period. Double treatment with both iNO and IV steroid tended to blunt the deterioration in renal function, although the only significant effect was on Base Excess (p = 0.045). None of the LPS + iNO + IV steroid treated animals died during the study period, whereas one animal died in each of the other LPS-infused groups. CONCLUSIONS: This study suggests that combined early therapy with iNO and IV steroid is associated with partial protection of kidney function after 30 hours of experimental LPS infusion

Development and validation of a simple LC-MS/MS method for simultaneous determination of moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol in human plasma

Treatment of multidrug-resistant tuberculosis (MDR-TB) is challenging due to high treatment failure rate and adverse drug events. This study aimed to develop and validate a simple LC-MS/MS method for simultaneous measurement of five TB drugs in human plasma and to facilitate therapeutic drug monitoring (TDM) in MDR-TB treatment to increase efficacy and reduce toxicity. Moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol were prepared in blank plasma from healthy volunteers and extracted using protein precipitation reagent containing trichloroacetic acid. Separation was achieved on an Atlantis T3 column with gradient of 0.1% formic acid in water and acetonitrile. Drug concentrations were determined by dynamic multiple reaction monitoring in positive ion mode on a LC-MS/MS system. The method was validated according to the United States' Food and Drug Administration (FDA) guideline for bioanalytical method validation. The calibration curves for moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol were linear, with the correlation coefficient values above 0.993, over a range of 0.1-5, 0.4-40, 0.2-10, 2-100 and 0.2-10 mg/L, respectively. Validation showed the method to be accurate and precise with bias from 6.5% to 18.3% for lower limit of quantification and -5.8% to 14.6% for LOW, medium (MED) and HIGH drug levels, and with coefficient of variations within 11.4% for all levels. Regarding dilution integrity, the bias was within 7.2% and the coefficient of variation was within 14.9%. Matrix effect (95.7%-112.5%) and recovery (91.4%-109.7%) for all drugs could be well compensated by their isotope-labelled internal standards. A benchtop stability test showed that the degradation of prothionamide was over 15% after placement at room temperature for 72 h. Clinical samples (n = 224) from a cohort study were analyzed and all concentrations were within the analytical range. The signal of prothionamide was suppressed in samples with hemolysis which was solved by sample dilution. As the method is robust and sample preparation is simple, it can easily be implemented to facilitate TDM in programmatic MDR-TB treatment

Population pharmacokinetics and model-based dosing evaluation of bedaquiline in multidrug-resistant tuberculosis patients

Aims: Bedaquiline is now recommended to all patients in the treatment of multidrug-resistant tuberculosis (MDR-TB) using standard dosing regimens. As the ability to measure blood drug concentrations is very limited, little is known about drug exposure and treatment outcome. Thus, this study aimed to model the population pharmacokinetics as well as to evaluate the currently recommended dosage.Methodology: A bedaquiline population pharmacokinetic (PK) model was developed based on samples collected from the development cohort before and 1, 2, 3, 4, 5, 6, 8, 12, 18, and 24 h after drug intake on week 2 and week 4 of treatment. In a prospective validation cohort of patients with MDR-TB, treated with bedaquiline-containing standardized regimen, drug exposure was assessed using the developed population PK model and thresholds were identified by relating to 2-month and 6-month sputum culture conversion and final treatment outcome using classification and regression tree analysis. In an exploratory analysis by the probability of target attainment (PTA) analysis, we evaluated the recommended dosage at different MIC levels by Middlebrook 7H11 agar dilution (7H11).Results: Bedaquiline pharmacokinetic data from 55 patients with MDR-TB were best described by a three-compartment model with dual zero-order input. Body weight was a covariate of the clearance and the central volume of distribution, albumin was a covariate of the clearance. In the validation cohort, we enrolled 159 patients with MDR-TB. The 7H11 MIC mode (range) of bedaquiline was 0.06 mg (0.008–0.25 mg/L). The study participants with AUC0-24h/MIC above 175.5 had a higher probability of culture conversion after 2-month treatment (adjusted relative risk, aRR:16.4; 95%CI: 5.3–50.4). Similarly, those with AUC0-24h/MIC above 118.2 had a higher probability of culture conversion after 6-month treatment (aRR:20.1; 95%CI: 2.9–139.4), and those with AUC0-24h/MIC above 74.6 had a higher probability of successful treatment outcome (aRR:9.7; 95%CI: 1.5–64.8). Based on the identified thresholds, simulations showed that the WHO recommended dosage (400 mg once daily for 14 days followed by 200 mg thrice weekly) resulted in PTA &gt;90% for the majority of isolates (94%; MICs ≤0.125 mg/L).Conclusion: We established a population PK model for bedaquiline in patients with MDR-TB in China. Based on the thresholds and MIC distribution derived in a clinical study, the recommended dosage of bedaquiline is sufficient for the treatment of MDR-TB

Real-time PCR detection of Human Herpesvirus 1-5 in patients lacking clinical signs of a viral CNS infection

<p>Abstract</p> <p>Background</p> <p>Infections of the central nervous system (CNS) with herpes- or enterovirus can be self-limiting and benign, but occasionally result in severe and fatal disease. The polymerase chain reaction (PCR) has revolutionized the diagnostics of viral pathogens, and by multiple displacement amplification (MDA) prior to real-time PCR the sensitivity might be further enhanced. The aim of this study was to investigate if herpes- or enterovirus can be detected in cerebrospinal fluid (CSF) from patients without symptoms.</p> <p>Methods</p> <p>Cerebrospinal fluid (CSF) samples from 373 patients lacking typical symptoms of viral CNS infection were analysed by real-time PCR targeting herpesviruses or enteroviruses with or without prior MDA.</p> <p>Results</p> <p>In total, virus was detected in 17 patients (4%). Epstein-Barr virus (EBV) was most commonly detected, in general from patients with other conditions (e.g. infections, cerebral hemorrhage). MDA satisfactorily amplified viral DNA in the absence of human nucleic acids, but showed poor amplification capacity for viral DNA in CSF samples, and did not increase the sensitivity for herpes virus-detection with our methodology.</p> <p>Conclusions</p> <p>Viral pathogens are rarely detected in CSF from patients without signs of CNS infection, supporting the view that real-time PCR is a highly specific method to detect symptomatic CNS-infection caused by these viruses. However, EBV may be subclinically reactivated due to other pathological conditions in the CNS.</p

Identification of recent tuberculosis exposure using QuantiFERON-TB Gold Plus, a multicenter study.

We investigated whether the difference of antigen tube 2 (TB2) minus antigen tube 1 (TB1) (TB22TB1) of the QuantiFERON-TB gold plus test, which has been postulated as a surrogate for the CD81 T-cell response, could be useful in identifying recent tuberculosis (TB) exposure. We looked at the interferon gamma (IFN-g) responses and differences in TB2 and TB1 tubes for 686 adults with QFT-plus positive test results. These results were compared among groups with high (368 TB contacts), low (229 patients with immune-mediated inflammatory diseases [IMID]), and indeterminate (89 asylum seekers or people from abroad [ASPFA]) risks of recent TB exposure. A TB22TB1 value .0.6 IU ml21 was deemed to indicate a true difference between tubes. In the whole cohort, 13.6%, 10.9%, and 11.2% of cases had a TB2.TB1 result in the contact, IMID, and ASPFA groups, respectively (P = 0.591). The adjusted odds ratios (aORs) for an association between a TB22TB1 result of .0.6 IU ml21 and risk of recent exposure versus contacts were 0.71 (95% confidence interval [CI], 0.31 to 1.61) for the IMID group and 0.86 (95% CI, 0.49 to 1.52) for the ASPFA group. In TB contact subgroups, 11.4%, 5.4%, and 17.7% with close, frequent, and sporadic contact had a TB2.TB1 result (P = 0.362). The aORs versus the close subgroup were 1.29 (95% CI, 0.63 to 2.62) for the frequent subgroup and 1.55 (95% CI, 0.67 to 3.60) for the sporadic subgroup. A TB22TB1 difference of .0.6 IU ml21 was not associated with increased risk of recent TB exposure, which puts into question the clinical potential as a proxy marker for recently acquired TB infection

Essays on Housing Deregulation and Investment Behavior

Welfare effects of deregulating the Swedish rental housing market The Swedish rental housing regulations have been rated the strictest among the OECD countries. In this chapter, I examine deregulatory reforms and how different redistributive schemes affect prices, inequality, and welfare. For this purpose, I build a static general equilibrium model with heterogeneous agents, which I calibrate to match the Swedish economy. I find that all households support a reform that allows households to freely trade rental contracts. Such a reform results in a welfare gain of 411 SEK per adult equivalent and year. How should rental housing deregulation be timed? I build on the first chapter and examine with what timing households would prefer that the rental housing market was deregulated. I develop a dynamic general equilibrium model of a small open economy. After calibrating the model to match salient features of the Swedish economy, I use it to test and evaluate four reforms that deregulate the rental market with different time profiles. I find that, in the long run, house prices and rents in the regulated housing stock increase by 267 and 25 percent, respectively, irrespective of the reform. In the unregulated housing stock, they fall by 19 and 6 percent, respectively. In the short run, nonhomeowner outsiders prefer reforms that remove regulations swiftly whereas homeowners and insiders prefer more gradual removals. The role of cognitive and noncognitive skills for investment behavior We analyze layman investment behavior in a mandatory defined contribution pension plan and find strong heterogeneity in behavior and performance outcomes. In the Swedish pension system reform of 2000, entrants in the year of the launch were 51 percentage points more likely to opt out from the default fund compared to those who entered one year later. Particularly likely to opt out were individuals with prior investment experience and high noncognitive skills. Cognitive skills, on the other hand, fostered activity in terms of reallocation between funds. As a consequence, the return loss associated with a one-standard deviation increase in noncognitive skills is estimated to 11 basis points per year while cognitive skills are unrelated to returns in the pension plan. We argue that the peculiar relationship between noncognitive skills and returns stems from the circumstances at the time of the launch—only pension investors who entered the plan in 2000 suffered a return loss associated with noncognitive skills—and that the correlation between noncognitive skills and opting out from the default fund at the launch of the reform is likely a result of the intense information and advertising campaigns that took place. Identity capital and wealth accumulation We develop a theory of identity capital. Identity is built up around a "life project" that can take many forms and in which individuals invest time and/or money; identity capital is a stock measure capturing these investments. In this paper, we focus on the life project of building a firm and its possible relevance for (1) the high propensity to save of rich entrepreneurs and (2) the rise of risky portfolio shares in wealth. To this end, we introduce identity capital into a dynamic consumption-savings model with uninsurable idiosyncratic risk. The key model feature is a utility asymmetry: We assume that decumulating, or losing, identity leads to a utility loss, while building it up renders no utility gain. We find that identity management makes individuals reluctant to downsize when the firm's financial prospects are weak as well as to invest when they are strong.  The model also implies that, at least in parts of the wealth-identity space, the risky portfolio share increases in wealth, a result that is hard to obtain in standard models with constant relative risk aversion

An implementation of capacity reservation devices in IP networks

pporting the project AMRAM, Dnr 2001-06251. Finally, I would like to thank my family, especially my mother for her unlimited support, and Miko Paues, my dear uncle and close friend, for proofreading my report at an early stage. i Contents 1 Introduction 1 1.1 Thesis introduction . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Task description . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.3 Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Quality of Service - Background 3 2.2 Service models . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2.1 Best eort . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2.2 Integrated Services - IntServ . . . . . . . . . . . . . . . 4 2.2.3 Dierentiated Services - DiServ . . . . . . . . . . . . 5 2.2.4 Discussion of the dierent service models . . . . . . . . 6 3 Bandwidth market 8 3.2 Participants of the secondary market . . . . . . . . . . . . . . 9 3.2.1 Market place . . . . . . . . . . . . .

Brain Stem Involvement in Immune and Aversive Challenge

Activation of the immune system by e.g. bacteria induces the acute-phase-response and sickness behaviour. The latter encompasses among other things fever, lethargy, anorexia and hyperalgesia. An often used model to study sickness behaviour is the intravenous injection of the gram negative bacterial endotoxin lipopolysaccharide (LPS). LPS induces the production of inflammatory mediators, such as cytokines and prostaglandins, which in turn can interact with the central nervous system (CNS) to affect behaviour. The CNS also memorises substances that have made us sick in the past to avoid future harm, a phenomenon called conditioned taste aversion (CTA). An often used model to study CTA is the intraperitoneal injection of LiCl. The pontine parabrachial nucleus (PB) is an autonomic relay nucleus situated in the rostral brain stem that integrates afferent somatosensory and interoceptive information and forwards this information to the hypothalamus and limbic structures. PB is crucial for the acquisition of CTA and PB neurons are activated by many anorexigenic substances. Further, PB neurons express neuropeptides, among those calcitonin gene related peptide (CGRP) and enkephalin, both of which have been implicated in immune signalling, nociception, food intake, and aversion. By using a dual-labelling immunohistochemical/in situ hybridization technique we investigated if enkephalinergic neurons in PB are activated by systemic immune challenge. While there were many neurons in the external lateral parabrachial subnucleus (PBel) that expressed the immediate early gene fos after intravenous injection of LPS and while a large proportion of the PBel neurons expressed preproenkephalin, there were very few double-labelled cells. The fos-expressing cells were predominantly located to the outer part of the PBel (PBelo), whereas the preproenkephalin-expressing PBel neurons were located closest to the peduncle. Thus we conclude that although enkephalin has been implicated in autonomic and immune signalling, enkephalinergic neurons in PB do not seem to be activated by immune stimulation (paper I). To further characterise the PBelo neurons activated by immune challenge we investigated if these neurons expressed CGRP. Dual-labelling in situ hybridisation showed that PBelo neurons that expressed fos after intravenous injection of LPS to a large extent co-expressed CGRP mRNA, indicating that CGRP may be involved in the regulation of the sickness response in immune challenge (paper II). Using dual-labelling immunohistochemistry we examined if PBel neurons activated by an immune stimulus projected to the amygdala, a limbic structure implicated in the affective response to homeostatic challenge. Animals were injected with the retrograde tracer substance cholera toxin b (CTb) into the amygdala and subsequently subjected to immune challenge. We found that approximately a third of the neurons that expressed fos after the intravenous injection of LPS also were labelled with CTb. Thus PBel neurons activated by immune challenge project to the amygdala. The PBel-amygdala pathway has earlier been suggested to be important in nociceptive signalling. To investigate if amygdala-projecting PBel neurons are activated by nociceptive stimuli we again injected animals with CTb into the amygdala. After recovery the animals were injected with formalin into a hindpaw. Dual-labelling immunohistochemistry against fos and CTb showed that very few noxiously activated PB neurons projected to the amygdala. Thus, the PBel-amygdala projection seems to be important in immune challenge but not in nociceptive signalling (paper III). Many PBel neurons express fos after intraperitoneal injection of LiCl. Melanocortins are neuropeptides that recently have been implicated in metabolism, food intake and aversive mechanisms. The PB is known to express melanocortin receptor-4 (MC4-R) mRNA. Using dual-labelling in situ hybridization we investigated if PB neurons activated by intravenous injection of LPS or intraperitoneal injection of LiCl expressed MC4-R mRNA. We found that many PBelo neurons were activated by either LPS or LiCl and that a large proportion of such activated neurons expressed MC4-R mRNA. Further, using dual-labelling in situ hybridization against MC4-R mRNA and CGRP mRNA, we found that a large proportion of the CGRP positive PBelo neurons also expressed MC4-R mRNA. In summary, this thesis shows that CGRP-expressing neurons in the PBel are activated by peripheral immune challenge, that lipopolysaccharide-activated PBel neurons project to the amygdala, that the amygdala-projecting neurons in the PBel are CGRP-positive, and that PBel neurons activated by immune or aversive challenge express MC4-R. Taken together, these data suggest the presence of a melanocortin-regulated CGRP-positive pathway from the PBel to the amygdala that relays information of importance to certain aspects of sickness behaviour.On the day of the defence date the title of article II was: Feeding-related immune responsive brain stem neurons: association with CGRP. Article II: Erratum for in Neuroreport 2001;12(16):inside back cover. Neuroreport 2001;12(13):inside back cover. Article III: Erratum in: J Comp Neurol. 2005; 483:489-90.</p