Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients.

RATIONALE: Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP. OBJECTIVES: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the ICU; to determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth. METHODS: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 min); isolation and culture of primary subglottic epithelial cells from control patients; laboratory analysis of host innate immune defenses. MEASUREMENTS AND MAIN RESULTS: Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic impairment was reversible on treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air-liquid interface model of primary human subglottic epithelium. CONCLUSIONS: Mechanical ventilation in the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage.

The enteropathogenic E. coli effector EspF targets and disrupts the nucleolus by a process regulated by mitochondrial dysfunction.

The nucleolus is a multifunctional structure within the nucleus of eukaryotic cells and is the primary site of ribosome biogenesis. Almost all viruses target and disrupt the nucleolus--a feature exclusive to this pathogen group. Here, using a combination of bio-imaging, genetic and biochemical analyses, we demonstrate that the enteropathogenic E. coli (EPEC) effector protein EspF specifically targets the nucleolus and disrupts a subset of nucleolar factors. Driven by a defined N-terminal nucleolar targeting domain, EspF causes the complete loss from the nucleolus of nucleolin, the most abundant nucleolar protein. We also show that other bacterial species disrupt the nucleolus, dependent on their ability to deliver effector proteins into the host cell. Moreover, we uncover a novel regulatory mechanism whereby nucleolar targeting by EspF is strictly controlled by EPEC's manipulation of host mitochondria. Collectively, this work reveals that the nucleolus may be a common feature of bacterial pathogenesis and demonstrates that a bacterial pathogen has evolved a highly sophisticated mechanism to enable spatio-temporal control over its virulence proteins.

FPR-1 is an important regulator of neutrophil recruitment and a tissue-specific driver of pulmonary fibrosis.

Neutrophils are the most abundant inflammatory cells at the earliest stages of wound healing and play important roles in wound repair and fibrosis. Formyl peptide receptor 1 (FPR-1) is abundantly expressed on neutrophils and has been shown to regulate their function, yet the importance of FPR-1 in fibrosis remains ill defined. FPR-1-deficient (fpr1-/-) mice were protected from bleomycin-induced pulmonary fibrosis but developed renal and hepatic fibrosis normally. Mechanistically, we observed a failure to effectively recruit neutrophils to the lungs of fpr1-/- mice, whereas neutrophil recruitment was unaffected in the liver and kidney. Using an adoptive transfer model we demonstrated that the defect in neutrophil recruitment to the lung was intrinsic to the fpr1-/- neutrophils, as C57BL/6 neutrophils were recruited normally to the damaged lung in fpr1-/- mice. Finally, C57BL/6 mice in which neutrophils had been depleted were protected from pulmonary fibrosis. In conclusion, FPR-1 and FPR-1 ligands are required for effective neutrophil recruitment to the damaged lung. Failure to recruit neutrophils or depletion of neutrophils protects from pulmonary fibrosis.

What are junior doctors for? The work of Foundation doctors in the UK: a mixed methods study.

OBJECTIVES: To examine what activities constitute the work of Foundation doctors and understand the factors that determine how that work is constructed. DESIGN: Cross-sectional mixed methods study. Questionnaire survey of the frequency with which activities specified in curricular documents are performed. Semistructured interviews and focus groups. SETTING: Postgraduate medical training in the UK. PARTICIPANTS: Doctors in their first 2 years of postgraduate practice (Foundation Programme). Staff who work with Foundation doctors-supervisors, nurses and employers (clinical; non-clinical). RESULTS: Survey data from 3697 Foundation doctors identified curricular activities (41/103, 42%) that are carried out routinely (performed at least once or twice per week by >75% of respondents). However, another 30 activities (29%) were carried out rarely (at least once or twice per week by <25% respondents), largely because they are routinely part of nurses', and not doctors', work. Junior doctors indicated their work constituted three roles: 'support' of ward and team, 'independent practitioner' and 'learner'. The support function dominated work, but conflicted with stereotyped expectations of what 'being a doctor' would be. It was, however, valued by the other staff groups. The learner role was felt to be incidental to practice, but was couched in a limited definition of learning that related to new skills, rather than consolidation and practice. Activities and perceived role were shaped by the organisational context, medical hierarchies and through relationships with nurses, which could change unpredictably and cause tension. Training progression did not affect what activities were done, but supported greater autonomy in how they were carried out. CONCLUSIONS: New doctors must be fit for multiple roles. Strategies for transition should manage graduates' expectations of real-world work, and encourage teams and organisations to better accommodate graduates. These strategies may help ensure that new doctors can adapt to the variable demands of the evolving multiprofessional workforce.

Hapten Synthesis, Antibody Development, and a Highly Sensitive Indirect Competitive Chemiluminescent Enzyme Immunoassay for Detection of Dicamba.

Although dicamba has long been one of the most widely used selective herbicides, some U.S. states have banned the sale and use of dicamba because of farmers complaints of drift and damage to nonresistant crops. To prevent illegal use of dicamba and allow monitoring of nonresistant crops, a rapid and sensitive method for detection of dicamba is critical. In this paper, three novel dicamba haptens with an aldehyde group were synthesized, conjugated to the carrier protein via a reductive-amination procedure and an indirect competitive chemiluminescent enzyme immunoassay (CLEIA) for dicamba was developed. The assay showed an IC50 of 0.874 ng/mL which was over 15 times lower than that of the conventional enzyme immunoassay. The immunoassay was used to quantify dicamba concentrations in field samples of soil and soybean obtained from fields sprayed with dicamba. The developed CLEIA showed an excellent correlation with LC-MS analysis in spike-and-recovery studies, as well as in real samples. The recovery of dicamba ranged from 86 to 108% in plant samples and from 105 to 107% in soil samples. Thus, this assay is a rapid and simple analytical tool for detecting and quantifying dicamba levels in environmental samples and potentially a great tool for on-site crop and field monitoring.

The locust jump: an integrated laboratory investigation.

The locust is well known for its ability to jump large distances to avoid predation. This class sets out a series of investigations into the mechanisms underlying the jump enabling students to bring together information from biomechanics, muscle physiology, and anatomy. The nature of the investigation allows it to be undertaken at a number of levels of complexity from relatively simple comparative observations to detailed analysis of the properties of the muscles and the energy storage systems involved in powering the jump. The relative size and robustness of the locust make it simple to handle and ideal for such investigations.

Tremor in the human hand following peripheral nerve transection and reinnervation.

Slow movements and position holding by the digits are both characterised by 8-10 Hz tremor which appears to be centrally generated. Denervation and subsequent reinnervation lead to significant alterations in peripheral connectivity and reflex organisation. We have tested the hypothesis that 8-10 Hz tremor is present in the digits of subjects following a complete nerve lesion. The frequency content of abduction and adduction movements was recorded in 12 index fingers and nine little fingers reinnervated subsequent to a complete ulnar nerve transection. An optical position laser transducer was used to measure digital movements, minimising mechanical interference to the system. Concurrently, surface electromyograms (EMG) were also recorded from first dorsal interosseus muscles (1DI) and abductor digiti minimi brevis (ADMB) muscles for index and little fingers, respectively. The maximal voluntary contraction (MVC) of the reinnervated muscles varied from 5.9% to 100% of those of the unimpaired, contralateral hands. The subjects performed abduction-adduction movements of the index and little fingers and a position holding task. Significant peaks in PSD curves of acceleration and rectified integrated EMG traces were identified. Tremor in the 8-10 Hz range was evident in both the acceleration and EMG signals for the majority of digits during both the slow movement and position holding tasks. These findings demonstrate the robust nature of these 8-10 Hz oscillations, even following the significant changes in peripheral connectivity of muscle and nerve resulting from nerve transection and reinnervation.

Prevalence and prediction of unrecognised diabetes mellitus and impaired glucose tolerance following acute stroke.

BACKGROUND: diabetes mellitus not only increases the risk of ischaemic stroke two- to four-fold but also adversely inXuences prognosis. The prevalence of recognised diabetes mellitus in acute stroke patients is between 8 and 20%, but between 6 and 42% of patients may have undiagnosed diabetes mellitus before presentation. Post-stroke hyperglycaemia is frequent and of limited diagnostic value and the oral glucose tolerance test assumes that the patient is clinically stable and eating normally. There is a need for a simple and reliable method to predict new diabetes mellitus in acute stroke patients. OBJECTIVES: to determine the prevalence of unrecognised diabetes mellitus and impaired glucose tolerance on hospital admission and 12 weeks later in acute stroke patients with post-stroke hyperglycaemia > or = 6.1 mmol/l. To measure the accuracy of hyperglycaemia and elevated glycosylated haemoglobin concentration in predicting the presence of unrecognised diabetes mellitus at 12 weeks. DESIGN: acute (<24 hours) stroke patients (cerebral infarction and primary intracerebral haemorrhage) with admission hyperglycaemia between 6.0 and 17 mmol/l and without a previous history of insulin-treated diabetes mellitus who were randomised into the Glucose Insulin in Stroke Trial between October 1997 and May 1999 were studied. The Glucose Insulin in Stroke Trial is a randomised controlled trial investigating the benefits of maintaining euglycaemia in acute stroke patients with mild to moderate hyperglycaemia. At 12 weeks, survivors underwent a 75 g oral glucose tolerance test. The positive predictive value and negative predictive value of admission plasma glucose > or = 6.1 mmol/l and elevated glycosylated haemoglobin concentration in predicting the presence of diabetes mellitus were used to estimate the prevalence of unrecognised diabetes mellitus in a consecutive series of 582 acute stroke admissions. RESULTS: 582 consecutive acute stroke patients were assessed for eligibility for the Glucose Insulin Stroke Trial, of whom 83 (14%) had recognised diabetes mellitus. One hundred and forty-two patients were randomised and 62 underwent a 3-month oral glucose tolerance test, of whom 26 (42%) had normal glucose tolerance, 23 (37%) had impaired glucose tolerance and 13 (21%) had diabetes mellitus. Admission plasma glucose > or = 6.1 mmol/l and glycosylated haemoglobin > or = 6.2% predicted the presence of previously unrecognised diabetes mellitus at 12 weeks with a positive predictive value of 80% and negative predictive value of 96%. The estimated prevalence of unrecognised diabetes mellitus in the total series of acute stroke admissions was 16-24%. CONCLUSIONS: one-third of all acute stroke patients may have diabetes mellitus. For patients presenting with post-stroke hyperglycaemia, impaired glucose tolerance or diabetes mellitus is present in two-thirds of survivors at 12 weeks. Admission plasma glucose > or = 6.1 mmol/l combined with glycosylated haemoglobin > or = 6.2% are good predictors of the presence of diabetes mellitus following stroke.