Memory CD4 T cell subsets are kinetically heterogeneous and replenished from naive T cells at high levels

Characterising the longevity of immunological memory requires establishing the rules underlying the renewal and death of peripheral T cells. However, we lack knowledge of the population structure and how self-renewal and de novo influx contribute to maintenance of memory compartments. Here, we characterise the kinetics and structure of murine CD4 T cell memory subsets by measuring the rates of influx of new cells and using detailed timecourses of DNA labelling that also distinguish the behaviour of recently divided and quiescent cells. We find that both effector and central memory CD4 T cells comprise subpopulations with highly divergent rates of turnover, and show that inflows of new cells sourced from the naive pool strongly impact estimates of memory cell lifetimes and division rates. We also demonstrate that the maintenance of CD4 T cell memory subsets in healthy mice is unexpectedly and strikingly reliant on this replenishment

Physiologically Based Simulations of Deuterated Glucose for Quantifying Cell Turnover in Humans.

In vivo [6,6-(2)H2]-glucose labeling is a state-of-the-art technique for quantifying cell proliferation and cell disappearance in humans. However, there are discrepancies between estimates of T cell proliferation reported in short (1-day) versus long (7-day) (2)H2-glucose studies and very-long (9-week) (2)H2O studies. It has been suggested that these discrepancies arise from underestimation of true glucose exposure from intermittent blood sampling in the 1-day study. Label availability in glucose studies is normally approximated by a "square pulse" (Sq pulse). Since the body glucose pool is small and turns over rapidly, the availability of labeled glucose can be subject to large fluctuations and the Sq pulse approximation may be very inaccurate. Here, we model the pharmacokinetics of exogenous labeled glucose using a physiologically based pharmacokinetic (PBPK) model to assess the impact of a more complete description of label availability as a function of time on estimates of CD4+ and CD8+ T cell proliferation and disappearance. The model enabled us to predict the exposure to labeled glucose during the fasting and de-labeling phases, to capture the fluctuations of labeled glucose availability caused by the intake of food or high-glucose beverages, and to recalculate the proliferation and death rates of immune cells. The PBPK model was used to reanalyze experimental data from three previously published studies using different labeling protocols. Although using the PBPK enrichment profile decreased the 1-day proliferation estimates by about 4 and 7% for CD4 and CD8+ T cells, respectively, differences with the 7-day and 9-week studies remained significant. We conclude that the approximations underlying the "square pulse" approach-recently suggested as the most plausible hypothesis-only explain a component of the discrepancy in published T cell proliferation rate estimates

A study of soil humic interactions with calcium phosphate and the development of a novel method of oxygen isotope analysis in orthophosphate

University of Technology, Sydney. Faculty of Science.There is increasing interest in limiting quantities of applied phosphorus fertilizer both for economic reasons and for environmental protection purposes and in facilitating the release of normally unavailable phosphorus for plant growth. The work presented in this thesis seeks to identify and understand some of the chemical reactions that occur between phosphate, calcium and soil humic acids. In addition, a novel electrospray ionisation mass spectrometric (ESI-MS) analytical method, which measures both highly enriched and natural abundance levels of oxygen isotopes in orthophosphate-soil systems, was developed and validated. The significance of this novel method is in its capability to provide an accurate, simple and sensitive means of tracing the sources and sinks of phosphate in soils as well as enabling an effective way to follow chemical reactions and their mechanisms. In soils, most of the phosphorus from fertilizers, if not washed away in runoff water, is converted to insoluble compounds, including calcium phosphates. Soil organic acids such as humic and fulvic acids may play an important role in influencing inorganic phosphate availability to plants by inhibiting the formation of thermodynamically stable calcium phosphates. Therefore, this work examines the formation of calcium phosphate phases in the presence of humic and fulvic acids extracted from soil sourced from the Sydney Basin in NSW, Australia. The combined techniques of pH-stat autotitration, Fourier transform infrared and laser Raman spectroscopy, as well as x-ray diffraction and elemental analyses facilitated this study. At 25°C under conditions of high supersaturation and a pH of 7.4 humic materials were found to delay calcium phosphate phase transformation processes. Under these conditions there was a delayed transformation of unstable amorphous calcium phosphate (ACP) to thermodynamically more stable octacalcium phosphate (OCP) and thence to an apatitic phase resembling poorly crystalline hydroxyapatite (HAp). Investigations at the lower pH of 5.7, and in the presence of humic acids, revealed that ACP was also precipitated initially, however humic-free solutions at this pH produced the metastable phase, dicalcium phosphate dihydrate (DCPD). ACP produced in the presence of humic materials persisted longer than DCPD in their absence, before ultimately hydrolyzing to OCP. Thus these results confirm that humic materials are geologically relevant inhibitors of calcium phosphate transformations and modify the availability of phosphate in soils by changing crystallisation behaviour from solution. The work presented here also aimed to study phosphate reactions in soil environments with the use of stable oxygen isotope labelling (¹⁸O) to monitor the change in orthophosphate oxygen isotopic distributions under acidic soil conditions. Current methods for determining oxygen isotope analysis in phosphate involve either lengthy procedures for conversion of the phosphate to carbon monoxide/dioxide, are insensitive, or are not amenable to rapid automated analyses. The ESI-MS analytical method developed in this thesis is rapid and can be readily applied to calcium phosphate materials, which are normally not amenable to this type of analysis. Humic-induced oxygen exchange was not detectable in ¹⁸O enriched calcium phosphate products that had been precipitated at pH 5.7 from solutions at 25°C or 80°C. Furnace temperatures of 600°C caused the solid-state delabelling of calcium phosphate products in the absence of soil humic material, however, delabelling was accentuated in its presence. The furnace-induced delabelling was also evident when KH₂PO₄ (solid) was subjected to the same conditions. The implications of these results are discussed in terms of the importance of considering extreme heat conditions, for example in the case of forest or bush fires or volcanic conditions, when assessing oxygen exchange processes that occur in terrestrial environments

Understanding Compliance Dynamics in Community Justice Settings

This article seeks to expand the existing literature on compliance in community justice settings by highlighting the importance of service user participation in efforts to achieve compliance. The article’s central argument is that although co-productive strategies can enhance service user participation, the degree to which co-production is achievable in penal supervision is perhaps uncertain, and has received insufficient theoretical or empirical attention. To address the gap in knowledge, the article draws on the data generated from a study of compliance in Wales, United Kingdom, and employs the Bourdieusian concepts of habitus, field, and capital to argue that the convergence of two key factors undermines the viability of co-productive strategies in penal settings. One factor is the service users’ habitus of powerlessness which may breed passivity rather than active participation. The second also relates to the power dynamics that characterize penal supervision contexts. Within these contexts, practitioners are statutorily empowered to implement and enforce the requirements of community orders. In the current target-focused policy climate in England and Wales, practitioners may prioritize measurable compliance over forms of compliance that stem from service user participation and engagement perhaps because these are not readily quantifiable

“What if the patient has a severe reaction, and it is my fault?” A qualitative study exploring factors for sustainable implementation of penicillin allergy delabelling

Background Penicillin allergy delabelling (PAD), the process of evaluating penicillin allergy labels, is a key target in antibiotic stewardship, but uptake of the procedure outside clinical studies is limited. We aimed to explore factors that need to be addressed to sustainably implement a clinical pathway for PAD. Methods We conducted a qualitative study based on semi-structured interviews with focus groups consisting of a purposive sample of twenty-five nurses and physicians working in four different hospitals in Western Norway. Systematic text condensation was applied for analysis. Results Psychological safety was reported as crucial for clinicians to perform PAD. A narrative of uncertainty and anticipated negative outcomes were negatively associated with PAD performance. Education, guidelines, and colleague- and leadership support could together create psychological safety and empower health personnel to perform PAD. Key factors for sustainable implementation of PAD were facilitating the informant’s profound motivation for providing optimal health care and for reducing antimicrobial resistance. Informants were motivated by the prospect of a simplified PAD procedure. We identified three main needs for implementation of PAD: (1) creating psychological safety; (2) utilising clinicians’ inherent motivation and (3) optimal organisational structures. Conclusion A planned implementation of PAD must acknowledge clinicians’ need for psychological safety and aid reassurance through training, leadership, and guidelines. To implement PAD as an everyday practice it must be minimally disruptive and provide a contextually adaptive logistic chain. Also, the clinician’s motivation for providing the best possible healthcare should be utilised to aid implementation. The results of this study will aid sustainable implementation of PAD in Norway. Ethics The study was approved by the Western Norway Regional Committee for Medical Research Ethics (Study No:199210).publishedVersio

Direct Single-Dose Drug-Provocation Test Is Safe for Delabelling Penicillin Low-Risk Reactions in Adults

Background: Penicillins (PENs) are the most frequent drug-allergic reactions trigger. However, diagnostic work-up is complex and time-consuming: it requires skin testing (ST) and drug-provocation test (DPT), needing faster delabelling strategies. Although direct DPT without previous STs has shown to be safe, most of the studies are performed in children or in North American, Asian, or Oceanian adults, with few studies in the European adult population. We explored its safety in European adult patients with low-risk PEN allergy history and, additionally, analysed ST role and T-cell involvement by lymphocyte trans- formation test (LTT). Methods: We prospectively evaluated > 16 years of PEN-allergic labelled patients referred to Málaga Regional University Hospital during 2023. They reported non-immediate reactions without alarm signs and unknown reactions. Direct-single-dose DPT was performed in all patients. If positive, ST and LTT were carried out after reaction resolution. Results: We included 269 patients with the culprits being an unidentified PEN (36%), amoxicillin (AX) (32%), and AX-clavulanic acid (AX- CLV) (31%); and the symptoms maculopapular exanthema (MPE) (34%) and unknown reaction during childhood (23%). Only 16 (5.9%) had positive DPT, being 56% for AX and 44% for AX- CLV, 81% developing MPE, none severe. Most DPT-reacting patients reported cutaneous non-immediate reactions in the index reaction, and only one had an unknown childhood reaction. The mean day interval between drug administration and symptom development was lower (p = 0.002) in positive DPT than in the index reaction (2 vs 5 days). Moreover, ST was positive in only 19% and LTTs in 86.7% of positive DPT patients. Conclusions: Direct-single-dose DPT is safe for delabelling PEN allergy in non-immediate reactions without alarm signs and unknown reactions. ST had a poor diagnostic value and LTT had a high one, confirming a T-cell involvement.This study has been supported by the Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofounded by European Regional Development Fund) (PI21/00329); and through its programme of Redes de Investigacion Cooperativa Orientadas al Resultado en Salud (RICORS): Enfermedades Inflamatorias (RD21/0002/0008). ML holds a contract from “Juan Rodes” program (JR23/0039) from the Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness [grants co-funded by European Social Fund (ESF)]. PDE holds a Sara Borrell research contract by Institute of Health “Carlos III” (CD22/00112). CM holds a “Nicolas Monardes” research contract by Andalusian Regional Ministry Health (RC- 0004-2021). Funding for open access charge: Universidad de Málaga/CBUA

Factors influencing implementation and adoption of direct oral penicillin challenge for allergy delabelling: a qualitative evaluation

Background: Over 95% of penicillin allergy labels are inaccurate and may be addressed in low-risk patients using direct oral penicillin challenge (DPC). This study explored the behaviour, attitudes and acceptability of patients, healthcare professionals (HCPs) and managers of using DPC in low-risk patients. // Methods: Mixed-method, investigation involving patient interviews and staff focus groups at three NHS acute hospitals. Transcripts were coded using inductive and deductive thematic analysis informed by the Theoretical Domains Framework. // Findings: Analysis of 43 patient interviews and three focus groups (28 HCPs: clinicians and managers) highlighted themes of ‘knowledge’, ‘beliefs about capabilities and consequences’, ‘environmental context’, ‘resources’, ‘social influences’, ‘professional role and identity’, ‘behavioural regulation and reinforcement’ and a cross-cutting theme of digital systems. Overall, study participants supported the DPC intervention. Patients expressed reassurance about being in a monitored, hospital setting. HCPs acknowledged the need for robust governance structures for ensuring clarity of roles and responsibilities and confidence. // Conclusion: There were high levels of acceptability among patients and HCPs. HCPs recognised the importance of DPC. Complexities of penicillin allergy (de)labelling were highlighted, and issues of knowledge, risk, governance and workforce were identified as key determinants. These should be considered in future planning and adoption strategies for DPC

Use of a penicillin allergy clinical decision rule to enable direct oral penicillin provocation: an international multicentre randomised control trial in an adult population (PALACE): study protocol

Introduction Penicillin allergies are highly prevalent in the healthcare setting and associated with the prescription of second-line inferior antibiotics. More than 85% of all penicillin allergy labels can be removed by skin testing and 96%–99% of low-risk penicillin allergy labels can be removed by direct oral challenge. An internally and externally validated clinical assessment tool for penicillin allergy, PEN-FAST, can identify a low-risk penicillin allergy without the need for skin testing; a score of less than 3 has a negative predictive value of 96.3% (95% CI, 94.1 to 97.8) for the presence of a penicillin allergy. It is hypothesised that PEN-FAST is a safe and effective tool for assessing penicillin allergy in an outpatient clinic setting. Methods and analysis This is an international, multicentre randomised control trial using the PEN-FAST tool to risk-stratify penicillin allergy labels in adult outpatients. The study’s primary objective is to evaluate the non-inferiority of using PEN-FAST score-guided management with direct oral challenge compared with standard care (defined as prick and intradermal skin testing followed by oral penicillin challenge). Participants will be randomised 1:1 to the intervention arm (direct oral penicillin challenge) or standard of care arm (skin testing followed by oral penicillin challenge, if skin testing is negative). The sample size of 380 randomised patients (190 per treatment arm) is required to demonstrate non-inferiority. Ethics and dissemination The study will be performed according to the guidelines of the Helsinki Declaration and is approved by the Austin Health Human Research Ethics Committee (HREC/62425/Austin-2020) in Melbourne Australia, Vanderbilt University Institutional Review Board (IRB #202174) in Tennessee, USA, Duke University Institutional Review Board (IRB #Pro00108461) in North Carolina, USA and McGill University Health Centre Research Ethics Board in Canada (PALACE/2022-7605). The results of this study will be published and presented in various scientific forums

Evaluating parental perspectives on pediatric antibiotic use following penicillin allergy delabeling

This DNP project sought to evaluate parental perspectives on PCN allergy delabeling in pediatric patients who completed a successful oral PCN challenge. This evaluation was performed at a pediatric primary care department of a Federally Qualified Health Center between the dates of July 5th, 2023, and December 8th, 2023. This project applied the Theory of Constraints by focusing on the first step: identify a system’s constraints (Goldratt & Cox, 2004). The desired outcome of this intervention was to better understand parental perspectives and their level of acceptance of their child’s PCN delabeled status after completing a PCN oral challenge. Through the use of phone surveys, created based on expert opinion and research by Vyles et al. (2018), Lanchover-Roth et al. (2019), and Bourke et al. (2015), we were able to survey 100% of participant’s parents (n = 17). Participants included pediatric patients who had completed a PCN oral challenge during the project’s timeline or within 1 year prior to the project’s initiation. Parents responded with overwhelming positivity to the surveys, with greater than 60% reporting they would be very comfortable with their child receiving PCN in the future and 100% of parents agreeing that their child was no longer allergic to PCN

Eliciting the barriers and enablers towards anaesthetists giving penicillin-based antibiotic prophylaxis to low-risk patients who have had their penicillin allergy label removed as part of a preoperative delabelling process

Background Some penicillin allergy labels can be removed by non-allergy specialists by direct oral challenge, but there is reluctance amongst anaesthetists to give penicillin to these patients. We aimed to assess anaesthetist beliefs about giving penicillin to patients delabelled by direct oral challenge. Methods A survey, developed using the Theoretical Domains Framework, was circulated to anaesthetists within a regional research network in England. Domains were rated using 5-point Likert scales. Overall and group medians were used to dichotomize domains rated by group into ‘relatively important/unimportant’ and ‘relative enabler/barrier’. Results We received 257 responses from six hospitals (response rate 49.7%). Seven domains were rated as important for all stakeholder groups and hospitals: Knowledge, Skills, Belief in Capabilities, Belief in Consequences, Memory/Attention/Decisions, Environmental Context and Resources, and Emotions. Social and Professional Role was also important to all respondents except those in one hospital. Intentions and Optimism were rated as important for some groups/hospitals and unimportant for others. All four other domains were rated as unimportant for all groups/hospitals. All domains rated as important were enablers for all groups/hospitals, with the exception of Memory/Attention/Decisions and Emotions, which were rated as discordant barriers/enablers between groups. This means they were acting as a barrier for some staff groups/hospitals and an enabler for others. Barrier domains (Reinforcement, Goals, Social Influences, Behavioural Regulation) were all rated unimportant. Conclusions Behavioural influences on giving penicillin prophylaxis to a delabelled patient are complex and nuanced. These findings could inform targeted interventions, both across and within hospitals and staff groups