Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor ...delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.
Adoptive cancer immunotherapy using chimeric antigen receptor (CAR) engineered T-cells holds great promise, although several obstacles hinder the efficient generation of cell products under good ...manufacturing practice (GMP). Patients are often immune compromised, rendering it challenging to produce sufficient numbers of gene-modified cells. Manufacturing protocols are labour intensive and frequently involve one or more open processing steps, leading to increased risk of contamination. We set out to develop a simplified process to generate autologous gamma retrovirus-transduced T-cells for clinical evaluation in patients with head and neck cancer. T-cells were engineered to co-express a panErbB-specific CAR (T1E28z) and a chimeric cytokine receptor (4αβ) that permits their selective expansion in response to interleukin (IL)-4. Using peripheral blood as starting material, sterile culture procedures were conducted in gas-permeable bags under static conditions. Pre-aliquoted medium and cytokines, bespoke connector devices and sterile welding/sealing were used to maximise the use of closed manufacturing steps. Reproducible IL-4-dependent expansion and enrichment of CAR-engineered T-cells under GMP was achieved, both from patients and healthy donors. We also describe the development and approach taken to validate a panel of monitoring and critical release assays, which provide objective data on cell product quality.
A number of recent reports published in the UK have put the quality of care of adults with Rheumatoid Arthritis (RA) centre stage. These documents set high standards for health care professionals and ...commissioning bodies that need to be implemented into routine clinical practice. We therefore have obtained the views of recipients and providers of care in inner city settings as to what they perceive are the barriers to providing integrated care.
We conducted focus groups and face to face interviews between 2005-8 with 79 participants (patients, carers, specialist medical and nursing outpatient staff and general practitioners (GPs)) working in or attending three hospitals and three primary care trusts (PCT).
Three barriers were identified that stood in the way of seamless integrated care in RA from the perspective of patients, carers, specialists and GPs: (i) early referral (e.g. 'gate keeper's role of GPs); (ii) limitations of ongoing care for established RA (e.g. lack of consultation time in secondary care) and (iii) management of acute flares (e.g. pressure on overbooked clinics).
This timely study of the multi-perspective views of recipients and providers of care was conducted during the time of publications of many important reports in the United Kingdom (UK) that highlighted key components in the provision of high quality care for adults with RA. To achieve seamless care across primary and secondary care requires organisational changes, greater personal and professional collaboration and GP education about RA.
Randomised controlled trials are the gold standard for testing whether a new intervention produces change in outcome measures but they do not assess underlying mechanisms of action. Process ...evaluation is an overarching framework for studying potential mechanisms in complex interventions but is rarely applied. Researchers and funders have increasingly called for the inclusion of process evaluations alongside randomised controlled trials of complex interventions but there is no standardised approach. This has led to wide variation in frameworks and methods used. Psychological interventions are particularly complex as they use the collaboration between patient and provider as the conduit for change. Many psychological interventions are being tested in healthcare settings for people with long-term conditions, such as type 2 diabetes. Patients with type 2 diabetes require intensive daily management and have many psychological problems, which contribute to sub optimal control. The clinical setting for this thesis is the D6 study, which tested whether a nurse led psychological intervention could improve glycaemic control in people with type 2 diabetes compared to an attention control condition. A process evaluation of D6 is conducted using a process evaluation framework derived from the literature.
Competencies in psychological techniques delivered by primary care nurses to support diabetes self-management were compared between the intervention and control arms of a cluster randomised ...controlled trial as part of a process evaluation. The trial was pragmatic and designed to assess effectiveness. This article addresses the question of whether the care that was delivered in the intervention and control trial arms represented high fidelity treatment and attention control, respectively.
Twenty-three primary care nurses were either trained in motivational interviewing (MI) and cognitive behavioural therapy (CBT) skills or delivered attention control. Nurses' skills in these treatments were evaluated soon after training (treatment arm) and treatment fidelity was assessed after treatment delivery for sessions midway through regimen (both arms) using the Motivational Interviewing Treatment Integrity (MITI) domains and Behaviour Change Counselling Index (BECCI) based on consultations with 151 participants (45% of those who entered the study). The MITI Global Spirit subscale measured demonstration of MI principles: evocation, collaboration, autonomy/support.
After training, median MITI MI-Adherence was 86.2% (IQR 76.9-100%) and mean MITI Empathy was 4.09 (SD 1.04). During delivery of treatment, in the intervention arm mean MITI Spirit was 4.03 (SD 1.05), mean Empathy was 4.23 (SD 0.89), and median Percentage Complex Reflections was 53.8% (IQR 40.0-71.4%). In the attention control arm mean Empathy was 3.40 (SD 0.98) and median Percentage Complex Reflections was 55.6% (IQR 41.9-71.4%).
After MI and CBT skills training, detailed assessment showed that nurses had basic competencies in some psychological techniques. There appeared to be some delivery of elements of psychological treatment by nurses in the control arm. This model of training and delivery of MI and CBT skills integrated into routine nursing care to support diabetes self-management in primary care was not associated with high competency levels in all skills.
ISRCTN75776892 ; date registered: 19/05/2010.
Suboptimal glycaemic control in type 2 diabetes (T2D) is common and associated with psychological barriers.
To investigate whether it was possible to train practice nurses in six psychological skills ...(Diabetes-6 D6) based on motivational interviewing (MI) and basic cognitive behaviour therapy (CBT), and whether integrating these with diabetes care was associated with improved glycaemic control over 18 months compared with standard care.
Two-arm, single-blind, parallel cluster randomised controlled trial in primary care.
Adult participants (
= 334) with T2D and persistent HbA1c ≥69.4 mmol/mol were randomised to receive 12 sessions of either the D6 intervention or standard care over 12 months. Practice nurses were trained in the six psychological skills and their competencies were measured by standardised rating scales. Primary outcome was a change in HbA1c level at 18 months from randomisation. Secondary outcomes were changes in systolic and diastolic blood pressure, body mass index, waist circumference, depressive symptoms, harmful alcohol intake, diabetes-specific distress, and cost-effectiveness.
Using intention-to-treat analysis, there was no significant difference between D6 intervention and standard care in HbA1c (mean difference -0.79 mmol/mol, 95% confidence interval CI = -5.75 to 4.18) or for any of the secondary outcomes. The competency level of D6 nurses was below the beginner proficiency level and similar to the standard-care nurses.
Training nurses in MI and basic CBT to support self-management did not lead to improvements in glycaemic control or other secondary outcomes in people with T2D at 18 months. It was also unlikely to be cost-effective. Furthermore, the increased contact with standard-care nurses did not improve glycaemic control.
The clinical potential of DNA and RNA-targeting therapeutics for airways disease has been hampered by the poor translation of promising drug candidates from cell culture to in vivo models and the ...clinic. For example, classical preclinical approaches routinely report 20-60% target knockdown effects in the lung, where 1 or 2 log effects are observed in isolated cell cultures in vitro. Preparation of monocellular suspensions of tissues by mechanoenzymatic disruption followed by cell sorting (TDCS) after in vivo drug dosing, however, can offer pharmacokinetic and pharmacodynamic insights on the effects of drugs to precise cell subpopulations. Moreover, this can be reliably achieved with up to 66% fewer animals than standard in vivo pharmacology approaches due to lower data variance afforded through analytics on defined, viable cell numbers. Here we describe the TDCS methodology for the isolation of total lung epithelia, lung macrophages, and epithelium/macrophage-depleted cell fractions from mouse lungs using a two-stage sorting process of immunomagnetic bead separation followed by flow cytometric sorting using fluorescent antibodies against well-established surface markers such as F4/80, CD11b, and CD326. Validated antibodies for additional cell types and markers are also provided.
Highlights • Training nurses in psychological skills can have a positive impact on patient care. • Nurses require appropriate support from their practice in order to use skills effectively. • ...Qualitative evaluation of psychological skills training can reveal important mechanisms that may contribute to translation into practice.
Abstract Objective This study examined the relationships between patients' beliefs about rheumatoid arthritis (using Leventhal's Common Sense Model) and their levels of disability, health-related ...quality of life, and disease activity. A proposed illness beliefs construct of “seriousness” was also investigated, combining beliefs about illness identity (symptoms), consequences, and timeline. Method A cross-sectional study evaluated 125 patients with rheumatoid arthritis from two South East London hospitals. Questionnaires assessed their illness beliefs, disability, and quality of life. An objective measure of disease activity was also obtained. Results Higher disability scores were associated with beliefs about identity ( r =.31, P <.01) and consequences ( r =.28, P <.01). Stronger control beliefs were associated with lower disability ( r =−.40, P <.01) and better physical ( r =.20, P <.05) quality of life. Disease activity scores, although positively related to disability scores ( r =.39, P <.01), showed no associations with illness beliefs. Multivariate analysis resulted in models accounting for 45.5%, 27.3%, and 19.3% variance in disability, “physical quality of life” scores, and “mental quality of life” scores, respectively. The hypothesis for a proposed “seriousness” construct was not supported. Conclusion Patients' beliefs about their rheumatoid arthritis are associated with disability and quality of life and cannot be explained by disease status. Longitudinal research is needed to assess the stability of beliefs.
BackgroundThe complement system plays a crucial part in innate immunity, aiding the destruction of pathogens and removing anomalous cells. Complement Factor H (CFH) regulates the complement system ...through interactions with C3b (both soluble and membrane associated) to prevent unwarranted activation. This forms the basis of a self-versus non-self-recognition process, which in certain cancers, aberrant expression, or abnormal levels of CFH may protect and promote growth of tumors by impairing the activation of the complement system. An analysis of human antibody repertoire data retrieved convergent sequence clusters amongst both viral and cancer (pancreatic, prostate and melanoma) patient cohorts, and led to identification of human derived antibodies that are naturally tolerated with unique properties1,2,3. A representative heavy chain from this cluster was paired with an appropriate light chain and engineered for desirable biophysical characteristics. Target identification revealed CFH, as a potential antigen.MethodsIn this study, we tested the ability of human derived antibodies to CFH to activate direct cell killing via complement deposition, and to alter immune cell responses (potentially via alterations in complement receptor activation). This was performed using human in vitro co-culture, immunomodulatory assays, and in vivo syngeneic mouse systems. Additionally, pharmacokinetics and associated measurements were assessed to understand basic tolerability and exposure.ResultsAnti-CFH resulted in increased CD11b+ cell tumor infiltration and subsequent C3d deposition in a B16/F10 syngeneic model. Macrophages adopted an inflammatory phenotype characterised by cytokine release profiles and lymphocyte activation.Immune cell co-culture with tumor cells resulted in myeloid activation, altered cell populations, indirect activation of additional immune cell types, and reduction in viable cancer cells. Both single and dose range pharmacokinetic studies confirmed a lack of binding to circulating CFH, and unaffected levels of C3 that might be associated with systemic complement activation. In a preliminary assessment, no safety issues were identified in the study. Monotherapy in high CFH syngeneic models, suggests a unique epitope recognised by anti-CFH which is primarily associated with membrane associated C3b. Interfering with this binding uniquely enables localised inflammation associated with the alternative pathway of the complement system. Additional mechanistic studies are ongoing.ConclusionsA human derived antibody cluster against CFH is convergently present in various disease state patient groups and has novel immunostimulatory function through engagement of myeloid cells and the complement cascade.ReferencesEhrhardt SA, et al. Polyclonal and convergent antibody response to Ebola virus vaccine rVSV-ZEBOV. Nat Med. 2019;25(10):1589–1600.Davis CW, et al. Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection. Cell. 2019;177(6):1566–1582.e17.Galson JD, et al. Deep Sequencing of B Cell Receptor Repertoires From COVID-19 Patients Reveals Strong Convergent Immune Signatures. Front Immunol. 2020;11:605170.
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