The Role of Bovine Cathelicidins in Host Defence

Cattle are constantly exposed to environmental pathogens and are susceptible to a number of diseases which cause significant economic loss or animal welfare concerns. The mucosal surface is a key barrier to infection and a greater understanding of bovine mucosal immunology may lead to improved disease management strategies. The cathelicidins are a family of host defence proteins which may play an important role in this defensive barrier. The cathelicidins are composed of a globular cathelin-like domain (CLD) and a C-terminal antimicrobial peptide (AMP) domain. The CLD is highly conserved across all species in which cathelicidins are found, yet the role of the CLD during infection has not been firmly established. The first aim of this thesis was to produce functional recombinant bovine CLD as a precursor to further experiments. However, the recombinant protein bound less lipopolysaccharide, was unable to agglutinate microbes, and was unable to permeabilise neutrophil membranes when compared to the activity of a native CLD preparation. Although further studies were not carried out with recombinant CLD, these results demonstrated that cell death induced by the native CLD and the agglutination of microbes potentially contribute to a broad anti-inflammatory role for the CLD during infection. In contrast to humans and mice where only one cathelicidin isoform is expressed, bovine express seven cathelicidins, with variable AMP domains. Therefore the second aim of this thesis was to profile the effect of bovine cathelicidin AMPs on neutrophil function. The bovine AMPs were able to modify a number of activities. Migration and reactive oxygen species (ROS) production were enhanced by several peptides while ROS production was inhibited by others. When investigated in further detail, linear Bac1 (bactenecin), Bac5 and BMAP-34 (bovine myeloid antimicrobial peptide) were able to dose-dependently induce or inhibit several key neutrophil functions including migration, degranulation, respiratory burst and phagocytosis, indicating significant roles in differential modulation of immune responses. In particular, Bac5 was able to differentially modify neutrophil respiratory burst without significant disruption to cellular homeostasis, which suggested Bac5 was acting via an intracellular mechanism. The third aim of this thesis was to investigate the mechanism by which Bac5 modulated neutrophil function. The results demonstrated the ability of Bac5 to be internalised by neutrophils and that Bac5 inhibition of p47phox binding to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a possible explanation for the differential induction and inhibition of extracellular ROS production. β-actin was identified as a major intracellular target for Bac5 and is consistent with the ability of Bac5 to modulate multiple neutrophil functions. In summary, this thesis demonstrates that the bovine cathelicidins have multiple roles in host defence. The conserved CLD appears to have an anti-inflammatory role through an as yet undefined mechanism. The variable AMPs together have multifaceted roles which may act in concert to promote elimination of pathogens and regulate excessive detrimental neutrophil activity. The combined effect of these roles will facilitate clearance of pathogens during infection and aid in the resolution of the innate inflammatory response at mucosal surfaces

Which children and young people are excluded from school? Findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) - poster abstract

Poster abstract presented at Spring Meeting for Clinician Scientists in Training 2015BACKGROUND: School exclusion is a disciplinary method used to remove a child from the school environment. It is known to affect certain groups disproportionately, including boys, some ethnic minorities, children in care, children in poverty, and children with special educational needs. Population-based studies on wider characteristics of excluded pupils are scarce. The aim of this study was to describe factors associated with school exclusion in the Avon Longitudinal Study of Parents and Children (ALSPAC), focussing on neurodevelopment and mental health. METHODS: ALSPAC is a prospective population-based British birth cohort study, with the initial sample consisting of 14 541 pregnancies. The study has data for whether a child has been permanently excluded from school up to the age of 8 years as reported by parents and also permanent and fixed period exclusions in the preceding 12 months as reported by parents and young people at age 16 years. Upstream risk factors were assessed for associations with exclusion on univariable analysis. The association with social communication difficulties was investigated with multivariable logistic regression. FINDINGS: Data for exclusions up to the age of 8 years were available for 8245 ALSPAC participants and 4482 participants for exclusion at age 16 years. 53 pupils (0·6%) were excluded from school by age 8 years, and 390 (8·7%) at age 16 years. The odds of exclusion by 8 years and at 16 years were increased with male sex (p=0·001 and p<0·0001, respectively), low family income (p=0·014 and p<0·0001), family adversity (p<0·0001 for both), maternal psychopathology (p=0·013 and p=0·004), low intelligence quotient (p=0·041 and p<0·0001), mental health difficulties (p<0·0001 for both), psychiatric disorder (p<0·0001 for both), social communication difficulties (p<0·0001 for both), antisocial activities (p=0·004 and p<0·0001), bullying or being bullied (p=0·005 and p<0·0001), low educational attainment (p<0·0001 for both), and increased special educational needs (p<0·0001 for both). On multivariable analysis, having social communication difficulties above a clinical threshold on the Social Communication Disorders Checklist was strongly associated with exclusion by 8 years (odds ratio 7·4, 95% CI 3·6-15·4) and at 16 years (2·3, 1·5-3·5), after adjustment for relevant confounders. INTERPRETATION: Although cohort attrition and small numbers of exclusions at 8 years are limitations, this study suggests that school exclusion is associated with numerous risk factors identifiable at or before primary school entry. Child health professionals have an important role in the holistic assessment of children who are excluded, or who are at risk of school exclusion. There is particular need to ensure that mental health and neurodevelopmental difficulties are appropriately recognised and supported. FUNDING: National Institute for Health Research Academic Clinical Fellowship

Highly selective and solvent-dependent reduction of Nitrobenzene to N-phenylhydroxylamine, azoxybenzene, and aniline catalyzed by phosphino-modified polymer immobilized ionic liquid-stabilized AuNPs

Gold nanoparticles stabilized by phosphine-decorated polymer immobilized ionic liquids (AuNP@PPh2-PIILP) is an extremely efficient multiproduct selective catalyst for the sodium borohydride-mediated reduction of nitrobenzene giving N-phenylhydroxylamine, azoxybenzene, or aniline as the sole product under mild conditions and a very low catalyst loading. The use of a single nanoparticle-based catalyst for the partial and complete reduction of nitroarenes to afford three different products with exceptionally high selectivities is unprecedented. Under optimum conditions, thermodynamically unfavorable N-phenylhydroxylamine can be obtained as the sole product in near quantitative yield in water, whereas a change in reaction solvent to ethanol results in a dramatic switch in selectivity to afford azoxybenzene. The key to obtaining such a high selectivity for N-phenylhydroxylamine is the use of a nitrogen atmosphere at room temperature as reactions conducted under an inert atmosphere occur via the direct pathway and are essentially irreversible, while reactions in air afford significant amounts of azoxy-based products by virtue of competing condensation due to reversible formation of N-phenylhydroxylamine. Ultimately, aniline can also be obtained quantitatively and selectively by adjusting the reaction temperature and time accordingly. Introduction of PEG onto the polyionic liquid resulted in a dramatic improvement in catalyst efficiency such that N-phenylhydroxylamine could be obtained with a turnover number (TON) of 100 000 (turnover frequency (TOF) of 73 000 h–1, with >99% selectivity), azoxybenzene with a TON of 55 000 (TOF of 37 000 h–1 with 100% selectivity), and aniline with a TON of 500 000 (TOF of 62 500 h–1, with 100% selectivity). As the combination of ionic liquid and phosphine is required to achieve high activity and selectivity, further studies are currently underway to explore whether interfacial electronic effects influence adsorption and thereby selectivity and whether channeling of the substrate by the electrostatic potential around the AuNPs is responsible for the high activity. This is the first report of a AuNP-based system that can selectively reduce nitroarenes to either of two synthetically important intermediates as well as aniline and, in this regard, is an exciting discovery that will form the basis to develop a continuous flow process enabling facile scale-up

Personal non-commercial use only

ABSTRACT. Objective. To implement a rheumatology department education retreat to systematically identify and address the key factors necessary to improve medical education in our division in preparation for developing a rheumatology academy. Methods. The Hospital for Special Surgery organized a retreat for the Rheumatology Department aimed at (1) providing formal didactics and (2) assessing participants&apos; self-reported skills and interest in education with the goal of directing this information toward formalizing improvement. In a mixed-methods study design, faculty and fellows in the Division of Rheumatology were surveyed online pre-and post-retreat regarding various aspects of the current education program, their teaching abilities, interest and time spent in teaching, divisional resources allocated, and how education is valued. Results. Enthusiasm for teaching was high before and rose further after the retreat. Confidence in abilities was higher than expected before but fell afterward. Many noted that the lack of specific feedback on teaching skills and useful metrics to assess performance prevented the achievement of educational excellence. Most responding felt lack of time, knowledge of how to teach well, and resources prevented them from making greater commitments to educational endeavors and participating fully and effectively in the department&apos;s teaching activities. Conclusion. While most rheumatology faculty members want to improve as teachers, they know neither where their educational strengths and weaknesses lie nor where or how to begin to change their teaching abilities. The key elements for an academy would thus be an educational environment that elevates the quality of teaching throughout the division and promotes teaching careers and education research, and raises the importance and quality of teaching to equivalence with clinical care and research. (J Rheumatol First Release April 15 2012

The Structural Basis for Promoter −35 Element Recognition by the Group IV σ Factors

The control of bacterial transcription initiation depends on a primary σ factor for housekeeping functions, as well as alternative σ factors that control regulons in response to environmental stresses. The largest and most diverse subgroup of alternative σ factors, the group IV extracytoplasmic function σ factors, directs the transcription of genes that regulate a wide variety of responses, including envelope stress and pathogenesis. We determined the 2.3-Å resolution crystal structure of the −35 element recognition domain of a group IV σ factor, Escherichia coli σ(E) (4), bound to its consensus −35 element, GGAACTT. Despite similar function and secondary structure, the primary and group IV σ factors recognize their −35 elements using distinct mechanisms. Conserved sequence elements of the σ(E) −35 element induce a DNA geometry characteristic of AA/TT-tract DNA, including a rigid, straight double-helical axis and a narrow minor groove. For this reason, the highly conserved AA in the middle of the GGAACTT motif is essential for −35 element recognition by σ(E) (4), despite the absence of direct protein–DNA interactions with these DNA bases. These principles of σ(E) (4)/−35 element recognition can be applied to a wide range of other group IV σ factors

Developing decision support tools incorporating personalised predictions of likely visual benefit versus harm for cataract surgery:research programme

Background Surgery for established cataract is highly cost-effective and uncontroversial, yet uncertainty remains for individuals about when to proceed and when to delay surgery during the earlier stages of cataract. Objective We aimed to improve decision-making for cataract surgery through the development of evidence-based clinical tools that provide general information and personalised risk/benefit information. Design We used a mixed methodology consisting of four work packages. Work package 1 involved the development and psychometric validation of a brief, patient self-reported measure of visual difficulty from cataract and its relief from surgery, named Cataract Patient-Reported Outcome Measure, five items (Cat-PROM5). Work package 2 involved the review and refinement of risk models for adverse surgical events (posterior capsule rupture and visual acuity loss related to cataract surgery). Work package 3 involved the development of prediction models for the Cat-PROM5-based self-reported outcomes from a cohort study of 1500 patients; assessment of the validity of preference-based health economic indices for cataract surgery and the calibration of these to Cat-PROM5; assessment of patients’ and health-care professionals’ views on risk–benefit presentation formats, the perceived usefulness of Cat-PROM5, the value of personalised risk–benefit information, high-value information items and shared decision-making; development of cataract decision aid frequently asked questions, incorporation of personalised estimates of risks and benefits; and development of a cataract decision quality measure to assess the quality of decision-making. Work package 4 involved a mixed-methods feasibility study for a fully powered randomised controlled trial of the use of the cataract decision aid and a qualitative study of discordant or mismatching perceptions of outcome between patients and health-care professionals. Setting Four English NHS recruitment centres were involved: Bristol (lead centre), Brighton, Gloucestershire and Torbay. Multicentre NHS cataract surgery data were obtained from the National Ophthalmology Database. Participants Work package 1 – participants (n = 822) were from all four centres. Work package 2 – electronic medical record data were taken from the National Ophthalmology Database (final set > 1M operations). Work package 3 – cohort study participants were from Bristol (n = 1200) and Gloucestershire (n = 300); qualitative and development work was undertaken with patients and health-care professionals from all four centres. Work package 4 – Bristol, Brighton and Torbay participated in the recruitment of patients (n = 42) for the feasibility trial and recruitment of health-care professionals for the qualitative elements. Interventions For the feasibility trial, the intervention was the use of the cataract decision aid, incorporating frequently asked questions and personalised estimations of both adverse outcomes and self-reported benefit. Main outcome measures There was a range of quantitative and qualitative outcome measures: questionnaire psychometric performance metrics, risk indicators of adverse surgical events and visual outcome, predictors of self-reported outcome following cataract surgery, patient and health-care practitioner views, health economic calibration measures and randomised controlled trial feasibility measures. Data sources The data sources were patient self-reported questionnaire responses, study clinical data collection forms, recorded interviews with patients and health-care professionals, and anonymised National Ophthalmology Database data. Results Work package 1 – Cat-PROM5 was developed and validated with excellent to good psychometric properties (Rasch reliability 0.9, intraclass correlation repeatability 0.9, unidimensionality with residual eigenvalues ≤ 1.5) and excellent responsiveness to surgical intervention (Cohen delta –1.45). Work package 2 – earlier risk models for posterior capsule rupture and visual acuity loss were broadly affirmed (C-statistic for posterior capsule rupture 0.64; visual acuity loss 0.71). Work package 3 – the Cat-PROM5-based self-reported outcome regression models were derived based on 1181 participants with complete data (R2 ≈ 30% for each). Of the four preference-based health economic indices assessed, two demonstrated reasonable performance. Cat-PROM5 was successfully calibrated to health economic indices; adjusted limited dependent variable mixture models offered good to excellent fit (root-mean-square error 0.10–0.16). The personalised quantitative risk information was generally perceived as beneficial. A cataract decision aid and cataract decision quality measure were successfully developed based on the views of patients and health-care professionals. Work package 4 – data completeness was good for the feasibility study primary and secondary variables both before and after intervention/surgery (data completeness range 100–88%). Considering ability to recruit, the sample size required, instrumentation and availability of necessary health economic data, a fully powered randomised controlled trial (patients, n = 800, effect size 0.2 standard deviations, power 80%; p = 0.05) of the cataract decision aid would be feasible following psychometric refinement of the primary outcome (the cataract decision quality measure). The cataract decision aid was generally well-received by patients and health-care professionals, with cautions raised regarding perceived time and workload barriers. Discordant outcomes mostly related to patient dissatisfaction, with no clinical problem found. Limitations The National Ophthalmology Database data are expected to include some errors (mitigated by large multicentre data aggregations). The feasibility randomised controlled trial primary outcome (the cataract decision quality measure) displayed psychometric imperfections requiring refinement. The clinical occurrence of discordant outcomes is uncommon and the study team experienced difficulty identifying patients in this situation. Future work Future work could include regular review of the risk models for adverse outcomes to ensure currency, and the technical precision of complex-numbers analysis of refractive outcome to invite opportunities to improve post-operative spectacle-free vision. In addition, a fully powered randomised controlled trial of the cataract decision aid would be feasible, following psychometric refinement of the primary outcome (the cataract decision quality measure); this would clarify its potential role in routine service delivery. Conclusions In this research programme, evidence-based clinical tools have been successfully developed to improve pre-operative decision-making in cataract surgery. These include a psychometrically robust, patient-reported outcome measure (Cat-PROM5); prediction models for patient self-reported outcomes using Cat-PROM5; prediction models for clinically adverse surgical events and adverse visual acuity outcomes; and a cataract decision aid with relevant general information and personalised risk/benefit predictions. In addition, the successful mapping of Cat-PROM5 to existing health economic indices was achieved and the performances of indices were assessed in patients undergoing cataract surgery. A future full-powered randomised controlled trial of the cataract decision aid would be feasible (patients, n = 800, effect size 0.2 standard deviations, power 80%; p = 0.05). Trial registration This trial is registered as ISRCTN11309852. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Programme Grants for Applied Research programme and will be published in full in Programme Grants for Applied Research; Vol. 10, No. 9. See the NIHR Journals Library website for further project information

Recurrence and mortality according to Estrogen Receptor status for breast cancer patients undergoing conservative surgery. Ipsilateral breast tumour recurrence dynamics provides clues for tumour biology within the residual breast

BACKGROUND: The study was designed to determine how tumour hormone receptor status affects the subsequent pattern over time (dynamics) of breast cancer recurrence and death following conservative primary breast cancer resection. METHODS: Time span from primary resection until both first recurrence and death were considered among 2825 patients undergoing conservative surgery with or without breast radiotherapy. The hazard rates for ipsilateral breast tumour recurrence (IBTR), distant metastasis (DM) and mortality throughout 10 years of follow-up were assessed. RESULTS: DM dynamics displays the same bimodal pattern (first early peak at about 24 months, second late peak at the sixth-seventh year) for both estrogen receptor (ER) positive (P) and negative (N) tumours and for all local treatments and metastatic sites. The hazard rates for IBTR maintain the bimodal pattern for ERP and ERN tumours; however, each IBTR recurrence peak for ERP tumours is delayed in comparison to the corresponding timing of recurrence peaks for ERN tumours. Mortality dynamics is markedly different for ERP and ERN tumours with more early deaths among patients with ERN than among patients with ERP primary tumours. CONCLUSION: DM dynamics is not influenced by the extent of conservative primary tumour resection and is similar for both ER phenotypes across different metastatic sites, suggesting similar mechanisms for tumour development at distant sites despite apparently different microenvironments. The IBTR risk peak delay observed in ERP tumours is an exception to the common recurrence risk rhythm. This suggests that the microenvironment within the residual breast tissue may enforce more stringent constraints upon ERP breast tumour cell growth than other tissues, prolonging the latency of IBTR. This local environment is, however, apparently less constraining to ERN cells, as IBTR dynamics is similar to the corresponding recurrence dynamics among other distant tissue