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

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 >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

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

Use of chest X-ray in the assessment of community acquired pneumonia in primary care - an intervention study

Objectives The aim of this study was to explore if consequent use of chest X-ray (CXR), when the physician is not sure of the diagnosis of pneumonia after clinical examination and CRP-testing, favors a more restrictive prescribing of antibiotics. Design This was an intervention study conducted between September 2015 and December 2017. Setting Two intervention primary health care centers (PHCCs) and three control PHCCs in the southeast of Sweden. Intervention All patients were referred for CXR when the physician s suspicion of pneumonia was unsure, or quite sure after CRP-testing. Control units managed patients according to their usual routine after clinical examination and CRP-testing. Subjects A total of 104 patients were included in the intervention group and 81 patients in the control group. The inclusion criteria of the study were clinically suspected pneumonia in patients &amp;gt;= 18 years, with respiratory symptoms for more than 24 h. Main outcome measure:Antibiotic prescribing rate. Results In the intervention group, 85% were referred for CXR and 69% were prescribed antibiotics, as compared to 26% and 77% in the control group. The difference in antibiotic prescribing rate was not statistically significant, unadjusted OR 0.68 [0.35-1.3] and adjusted OR 1.1 [CI 0.43-3.0]. A total of 24% of patients with negative CXR were prescribed antibiotics. Conclusion This study could not prove that use of CXR when the physician was not sure of the diagnosis of pneumonia results in lowered antibiotic prescribing rate in primary care. In cases of negative findings on CXR the physicians do not seem to rely on the outcome when it comes to antibiotic prescribing

DNA methylome-based validation of induced sputum as an effective protocol to study lung immunity : construction of a classifier of pulmonary cell types

Flow cytometry is a classical approach used to define cell types in peripheral blood. While DNA methylation signatures have been extensively employed in recent years as an alternative to flow cytometry to define cell populations in peripheral blood, this approach has not been tested in lung-derived samples. Here, we compared bronchoalveolar lavage with a more cost-effective and less invasive technique based on sputum induction and developed a DNA methylome-based algorithm that can be used to deconvolute the cell types in such samples. We analysed the DNA methylome profiles of alveolar macrophages and lymphocytes cells isolated from the pulmonary compartment. The cells were isolated using two different methods, sputum induction and bronchoalveolar lavage. A strong positive correlation between the DNA methylome profiles of cells obtained with the two isolation methods was found. We observed the best correlation of the DNA methylomes when both isolation methods captured cells from the lower parts of the lungs. We also identified unique patterns of CpG methylation in DNA obtained from the two cell populations, which can be used as a signature to discriminate between the alveolar macrophages and lymphocytes by means of open-source algorithms. We validated our findings with external data and obtained results consistent with the previous findings. Our analysis opens up a new possibility to identify different cell populations from lung samples and promotes sputum induction as a tool to study immune cell populations from the lung.Funding Agencies|Forskningsradet Sydostra Sverige [FORSS-932096]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2015-02593, 2018-02961]; Swedish Heart Lung FoundationSwedish Heart-Lung Foundation [20150709, 20180613]; Medical Infection and Inflammation Center (MIIC) at Linkoping University; Forskningsradet i Sydostra Sverige [FORSS-932096]; Hjart-Lungfonden [20150709, 20180613]; VetenskapsradetSwedish Research Council [2015-02593, 2018-02961]</p

The spectrum of tuberculosis described as differential DNA methylation patterns in alveolar macrophages and alveolar T cells

Background: Host innate immune cells have been identified as key players in the early eradication of Mycobacterium tuberculosis and in the maintenance of an anti-mycobacterial immune memory, which we and others have shown are induced through epigenetic reprogramming. Studies on human tuberculosis immunity are dominated by those using peripheral blood as surrogate markers for immunity. We aimed to investigate DNA methylation patterns in immune cells of the lung compartment by obtaining induced sputum from M. tuberculosis- exposed subjects including symptom-free subjects testing positively and negatively for latent tuberculosis as well as patients diagnosed with active tuberculosis. Alveolar macrophages and alveolar T cells were isolated from the collected sputum and DNA methylome analyses performed (Illumina Infinium Human Methylation 450 k).Results: Multidimensional scaling analysis revealed that DNA methylomes of cells from the tuberculosis-exposed subjects and controls appeared as separate clusters. The numerous genes that were differentially methylated between the groups were functionally connected and overlapped with previous findings of trained immunity and tuberculosis. In addition, analysis of the interferon-gamma release assay (IGRA) status of the subjects demonstrated that the IGRA status was reflected in the DNA methylome by a unique signature.Conclusions: This pilot study suggests that M. tuberculosis induces epigenetic reprogramming in immune cells of the lung compartment, reflected as a specific DNA methylation pattern. The DNA methylation signature emerging from the comparison of IGRA-negative and IGRA-positive subjects revealed a spectrum of signature strength with the TB patients grouping together at one end of the spectrum, both in alveolar macrophages and T cells. DNA methylation-based biosignatures could be considered for further development towards a clinically useful tool for determining tuberculosis infection status and the level of tuberculosis exposure.Funding Agencies|Linkoeping University; Forskningsradet Sydoestra Sverige; Swedish Research Council [FORSS-932096]; Swedish Heart Lung Foundation [2015-02593, 2018-02961, 2018-04246, 106-2018-FONDECYT]; CONCYTEC-PROCIENCIA [2018-05973, 20150709]; Board of Research at the Karolinska Institute, Stockholm [20180613]; World Infection Fund</p

Plasma Levels of Rifampin Correlate with the Tuberculosis Drug Activity Assay

The plasma tuberculosis drug activity (TDA) assay may be an alternative tool for therapeutic drug monitoring in resource-limited settings. In tuberculosis (TB) patients (n = 30), TDA and plasma levels of first-line drugs were analyzed 2 h post-dose, 2 weeks after treatment initiation. Patients with plasma levels of rifampin lower than 8 mg/liter had a significantly lower median TDA (1.40 versus 1.68, P = 0.0013). TDA may be used to identify TB patients with suboptimal rifampin levels during TB treatment.Funding Agencies|Research Council of Southeast Sweden (FORSS); Marianne and Marcus Wallenberg Foundation; Swedish Heart and Lung Foundation; Region of Ostergotland, Sweden; Swedish Research Council</p