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Researchers from the WIMM kick off a week-long science extravaganza today at The Royal Society Summer Science Exhibition. The team will be exhibiting a unique blend of genetics and virtual reality on their stand, ‘DNA Origami: How do you fold a genome?’
The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks.
The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.
Development of a best-practice clinical guideline for the use of bleomycin in the treatment of germ cell tumours in the UK.
Bleomycin, a cytotoxic chemotherapy agent, forms a key component of curative regimens for lymphoma and germ cell tumours. It can be associated with severe toxicity, long-term complications and even death in extreme cases. There is a lack of evidence or consensus on how to prevent and monitor bleomycin toxicity. We surveyed 63 germ cell cancer physicians from 32 cancer centres across the UK to understand their approach to using bleomycin. Subsequent guideline development was based upon current practice, best available published evidence and expert consensus. We observed heterogeneity in practice in the following areas: monitoring; route of administration; contraindications to use; baseline and follow-up investigations performed, and advice given to patients. A best-practice clinical guideline for the use of bleomycin in the treatment of germ cell tumours has been developed and includes recommendations regarding baseline investigations, the use of pulmonary function tests, route of administration, monitoring and patient advice. It is likely that existing heterogeneity in clinical practice of bleomycin prescribing has significant economic, safety and patient experience implications. The development of an evidence-based consensus guideline was supported by 93% of survey participants and aims to address these issues and homogenise practice across the UK.
Nitrite levels in breath condensate of CF patients are raised in contrast to exhaled Nitric Oxide (NO) levels
NO is released by activated macrophages, neutrophils and epithelial cells in vitro and is elevated in the exhaled breath of asthmatics. Exhaled NO has thus been put forward as a marker of airways inflammation. However, we have found that, in contrast to asthma, exhaled NO is not elevated in the inflammatory airways diseases of bronchiectasis and cystic fibrosis (CF)1. We, therefore, questioned if exhaled NO reflected an accurate measure of NO production in the airways, particularly in chronically suppurative airways. We explored this by measuring nitrite, a stable end product of NO oxidation in breath condensate as a surrogate marker of NO production. Methods: 1.5 mls of breath condensate were collected over 6 minutes with subjects exhaling from total lung capacity against a resistance created by 0.5 mm diameter Teflon tubing immersed in ice. Nitrite was assayed using the Griess reaction. 21 clinically stable CF patients (mean age 25.4 y; mean FEV1 53.9%) and 11 normal subjects (mean age 28.8) were studied. Nitrite levels in breath condensate were then compared to exhaled NO levels, measured using the chemiluminescence analyser (Logan Research), and FEV1. Results: We found nitrite levels in CF patients to be significantly higher than normal subjects - median: 1.93 μM v 0.33 μM, p<0.001 by Mann Whitney Rank sum test. In contrast their exhaled NO values were not significantly different from normals1. There was no correlation between nitrite levels and exhaled NO or nitrite and FEV1. (Graph Presented). Conclusion: These results suggest that exhaled NO may not reflect total NO production, particularly in chronically suppurative airways. Exhaled NO does not give an indication of the source of NO production,. Possibly, the majority of NO is produced by inflammatory cells rather than epithelial cells and reacts close to its site of production and therefore not detected as free gas in the breath.
The respiratory epithelium is the major interface between the environment and the host. Sophisticated barrier, sensing, anti-microbial and immune regulatory mechanisms have evolved to help maintain homeostasis and to defend the lung against foreign substances and pathogens. During influenza virus infection, these specialised structural cells and populations of resident immune cells come together to mount the first response to the virus, one which would play a significant role in the immediate and long term outcome of the infection. In this review, we focus on the immune defence machinery of the respiratory epithelium and briefly explore how it repairs and regenerates after infection.
Longitudinal COVID-19 profiling associates IL-1RA and IL-10 with disease severity and RANTES with mild disease.
BACKGROUND: Identifying immune correlates of COVID-19 disease severity is an urgent need for clinical management, vaccine evaluation, and drug development. Here, we present a temporal analysis of key immune mediators, cytokines, and chemokines in blood of hospitalized COVID-19 patients from serial sampling and follow-up over 4 weeks. METHODS: A total of 71 patients with laboratory-confirmed COVID-19 admitted to Beijing You'an Hospital in China with either mild (53 patients) or severe (18 patients) disease were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines, and chemokines in peripheral blood every 4-7 days over 1 month per patient using a bioplex multiplex immunoassay. RESULTS: We found that the chemokine RANTES (CCL5) was significantly elevated, from an early stage of the infection, in patients with mild but not severe disease. We also found that early production of inhibitory mediators including IL-10 and IL-1RA were significantly associated with disease severity, and a combination of CCL5, IL-1 receptor antagonist (IL-1RA), and IL-10 at week 1 may predict patient outcomes. The majority of cytokines that are known to be associated with the cytokine storm in virus infections such as IL-6 and IFN-γ were only significantly elevated in the late stage of severe COVID-19 illness. TNF-α and GM-CSF showed no significant differences between severe and mild cases. CONCLUSION: Together, our data suggest that early intervention to increase expression of CCL5 may prevent patients from developing severe illness. Our data also suggest that measurement of levels of CCL5, as well as IL-1RA and IL-10 in blood individually and in combination, might be useful prognostic biomarkers to guide treatment strategies.
Idiopathic pulmonary fibrosis (IPF) is the most severe form of lung fibrosis. It is progressive, and has an extremely poor outcome and limited treatment options. The disease exclusively affects the lungs, and thus less attention has been focused on blood-borne immune cells. which could be a more effective therapeutic target than lung-based cells. Here, we questioned if circulating monocytes, which has been shown to be increased in IPF, bore abnormalities that might contribute to its pathogenesis. We found that levels of circulating monocytes correlated directly with the extent of fibrosis in the lungs, and increased further during acute clinical deterioration. Monocytes in IPF were phenotypically distinct, displaying increased expression of CD64, a type 1 IFN gene expression signature and a greater magnitude of type 1 IFN response when stimulated. These abnormalities were accompanied by markedly raised CSF-1 levels in the serum, prolonged survival of monocytes ex vivo, and increased numbers of monocytes in lung tissue. Our study defines the key monocytic abnormalities in IPF, proposing type 1 IFN-primed monocytes as a potential driver of an aberrant repair response and fibrosis. It provides a rationale for targeting monocytes and identifies monocytic CD64 as a potential specific therapeutic target for IPF.
The human lung is constantly exposed to the environment and potential pathogens. As the interface between host and environment, the respiratory epithelium has evolved sophisticated sensing mechanisms as part of its defense against pathogens. In this review, we examine how the respiratory epithelium senses and responds to influenza A virus, the biggest cause of respiratory viral deaths worldwide.
Exhaled nitric oxide is decreased by bronchoconstriction: An effect of airflow velocity in the airways
Exhaled NO in asthmatics is raised compared to normal subjects and it has been suggested that this measurement may be used as a non invasive method to monitor airway inflammation or response to treatment. We have noted annecdotally that patients with acute exacerbation of athma did not have substantially higher levels of exhaled NO. We therefore hypothesized that exhaled NO levels using currently accepted methods may be affected by the diameter of the airways. Methods: 10 steroid naive asthmatics (mean age 42y, mean FEV1 93%) and 10 randomly chosen asthmatics from our asthma database (on variable amounts of inhaled steroids, mean FEV1 83%, mean age 40.4y) were recruited. For the first group, exhaled NO was measured and FEV1 performed on the same day. For the second 10, exhaled NO was measured before and after histamine challenge (immediately after reaching PC20). In 4 of the 10, exhaled NO was also measured after FEV1 returned to basal levels with nebulised Salbutamol. NO was measured using single exhalation method with a chemiluminscence analyser (LR2000) at a constant flow rate and mouth pressure. Results: There was a strong corrleation between absolute FEV1 (but not % predicted FEV1) and exhaled NO in the steroid naive asthmatics (r=0.9, p<0.001, Pearson Product Moment Correlation test). In the second group, mere was a significant decrease in exhaled NO after histamine challenge (mean: 14.7 ppb (pre) v 11.6 ppb (post), p=0.001, paired t-test). There was decrease in NO in all subjects. In the 4 subjects who received a bronchodilator, exhaled NO returned to pre histamine challenge levels. Conclusion: The diameter of the airways affects the level of exhaled NO. This could be because the method of NO analysing requires a constant exhalation flow rate in all subjects in order to standardise measurements. In order to achieve the same flow rate, air flow velocity in narrower airways is increased and thus greater than that in the wider airways. For the faster airflow, there is less opportunity to remove NO produced by the airways, resulting in lower concentration of NO in the exhaled air. Thus, NO levels in exhaled breath of conditions affected by change in airways diameter may not reflect NO production in the lower airways. This may confound the use of exhaled NO as a marker of airways inflammation in asthma.
Exhaled Nitric Oxide (NO) is not elevated in patients with cystic fibrosis or bronchiectasis compared to normals
Airways inflammation has been associated with increased NO in the exhaled breath (Barnes, ERJ (6), 1993). We therefore questioned whether exhaled NO could act as an indicator of the severity of airways inflammation in the suppurative lung diseases, cystic fibrosis (CF) and bronchiectasis (Br). Methods: Single exhalation NO levels were measured using the chemiluminescence NO analyser (Logan Research, Kent); detection limit 0.3ppb. Upper airway NO production was excluded by maintaining a constant mouth pressure (4-5 cm H2O) during exhalation. This pushes back the soft palate and excludes air originating from the nose during exhalation. Values quoted are plateau levels at the end of exhalation, against a flow restrictor. Patients. All patients were recruited from our outpatient clinics. All except 3 CF patients have fully defined genotypes. Br was diagnosed by CT or classical CXR changes. All subjects were non smokers. 6 asthmatics were on inhaled steroids. All levels were taken when patients were stable. Results: normal CF Br Br+ster Asthma n=17 n=31 n=6 n=5 n=7 NO 5.5 (3.6) 4.5(2.6) 6.6(3.4) 5.9(1.4) 15.0(6.1) Age 32.8(16.1) 27.2(8.7) 49.3(8.4) 57.8(13.3) 39.6(16.3) FEV1 - 54.4(24.1) 61.0(22.8) 57.8(13.3) (All values are mean (SD), NO in ppb. Br= Bronchiectasis, Ster= inhaled fluticasone, budesonide or beclomethasone, FEV1 = % predicted.) These data suggest that NO in the exhaled breath of patients with CF and Br, in contrast to asthma, are not elevated. Explanations include a) poor diffusion of NO across increased and viscous airway secretion b) down regulation of iNOS in chronic suppurative conditions. Our findings in bronchiectasis contrast with those of Kharitonov et al (AJRCCM (151) 1995). This may be due to different methodologies.