RESUMO
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 (4αß) 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.
Assuntos
Citocinas/metabolismo , Interleucina-4/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Transdução GenéticaRESUMO
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.
Assuntos
Citometria de Fluxo , Pulmão/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Células Epiteliais Alveolares/metabolismo , Animais , Biomarcadores , Separação Imunomagnética/métodos , Pulmão/citologia , Pulmão/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos , Oligonucleotídeos/uso terapêutico , Especificidade de ÓrgãosRESUMO
BACKGROUND: Suboptimal glycaemic control in type 2 diabetes (T2D) is common and associated with psychological barriers. AIM: 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. DESIGN AND SETTING: Two-arm, single-blind, parallel cluster randomised controlled trial in primary care. METHOD: Adult participants (n = 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Glicemia/análise , Terapia Cognitivo-Comportamental , Diabetes Mellitus Tipo 2/enfermagem , Diabetes Mellitus Tipo 2/psicologia , Entrevista Motivacional , Atenção Primária à Saúde , Adulto , Idoso , Análise por Conglomerados , Análise Custo-Benefício , Depressão/complicações , Depressão/enfermagem , Depressão/psicologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Inglaterra , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Comportamento de Redução do Risco , Autogestão/métodos , Método Simples-Cego , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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%). CONCLUSIONS: 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. TRIAL REGISTRATION: ISRCTN75776892 ; date registered: 19/05/2010.
Assuntos
Competência Clínica , Terapia Cognitivo-Comportamental , Diabetes Mellitus Tipo 2/terapia , Educação Continuada em Enfermagem , Entrevista Motivacional , Enfermagem de Atenção Primária , Adulto , Terapia Cognitivo-Comportamental/educação , Aconselhamento/educação , Feminino , Humanos , Pessoa de Meia-Idade , AutogestãoRESUMO
AIM: Evidence for the efficacy of psychological skills training as a method of supporting patients' self-management is growing, but there is a shortage of mental health providers with specialist diabetes knowledge to deliver them. Primary care nurses are now increasingly expected to learn and use these techniques. This study explores nurse experience of training in six psychological skills to support patients' self-management of type 2 diabetes. METHODS: Semi-structured interviews elicited themes relating to nurses' experiences of participating in a trial of a psychological intervention, the Diabetes-6 study (D-6). Nurses were employed in GP surgeries in 5 South London boroughs. Thematic framework analysis was used to compare and contrast themes across participants. Nine nurses delivering the intervention (n=11), and 7 from the control intervention (n=12, no psychological element) were interviewed. RESULTS: Three key themes were identified: (i) positive and negative impact of D6 on nurses' practice: positives included patient empowerment; negatives included patients' capacity to engage; (ii) professional boundaries including concerns about over-stepping role as a nurse and (iii) concerns about degree of support from physicians at participating practices in integrating psychological and diabetes care. CONCLUSION: Primary care nurses report that psychological skills training can have a positive impact on patient care. Significant role adjustment is required, which may be aided by additional support from the practice team. Qualitative evaluation of effectiveness of psychological interventions may reveal processes that hinder or contribute to efficacy and translation. Appropriate support is necessary for primary care nurses to deliver psychological therapies with confidence.
Assuntos
Diabetes Mellitus Tipo 2/enfermagem , Educação Continuada em Enfermagem , Capacitação em Serviço , Entrevista Motivacional , Papel do Profissional de Enfermagem , Enfermagem de Atenção Primária , Autocuidado , Agendamento de Consultas , Atitude do Pessoal de Saúde , Terapia Cognitivo-Comportamental , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Entrevistas como Assunto , Londres , Pessoa de Meia-Idade , Relações Enfermeiro-Paciente , Educação de Pacientes como Assunto , Participação do Paciente , Pesquisa QualitativaRESUMO
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.
Assuntos
Apolipoproteínas B/antagonistas & inibidores , Inativação Gênica , Luciferases/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos/farmacocinética , RNA Interferente Pequeno/genética , Administração por Inalação , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Células Cultivadas , Marcação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Distribuição TecidualRESUMO
We assessed the inter-relationships between the Short Form 36 (SF-36) physical and mental function in 220 patients with onset cases of mild and moderate depression and 913 adults with early and established rheumatoid arthritis (RA) through secondary analysis and compared both scores with the UK general population norms. In depression and RA the SF-36 total scores showed significant impairment across the spectrum of both domains compared with age-specific UK normative score. In RA mental health and role, mental scores were highly correlated with other SF-36 domains. In depression there was little evidence of such inter-relationships. Mental health and role mental domains were lowest in active RA (disease activity scores (DAS28) over 5.1). They had strong correlations with the vitality and social function SF-36 sub-scores and weak correlations with the physical function and role emotional sub-scores. Patients with long-term conditions require comprehensive care. At present it is unclear how best to combine treatment of RA synovitis with the management of mental health problems. Mental health symptoms are present from the earliest stages of RA and it may be appropriate to initiate multidisciplinary care as soon as practicable, although its efficacy requires a further detailed study across primary and secondary care.
Assuntos
Artrite Reumatoide/psicologia , Transtorno Depressivo/psicologia , Qualidade de Vida/psicologia , Adulto , Análise de Variância , Distribuição de Qui-Quadrado , Feminino , Humanos , Masculino , Saúde Mental , Pessoa de Meia-Idade , Ajustamento Social , Inquéritos e Questionários , Reino UnidoRESUMO
BACKGROUND: 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. METHODS: 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). RESULTS: 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). CONCLUSION: 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.
Assuntos
Artrite Reumatoide/terapia , Pessoal de Saúde/tendências , Acessibilidade aos Serviços de Saúde/tendências , Serviços Urbanos de Saúde/tendências , População Urbana/tendências , Feminino , Grupos Focais , Clínicos Gerais/organização & administração , Clínicos Gerais/tendências , Pessoal de Saúde/organização & administração , Acessibilidade aos Serviços de Saúde/organização & administração , Humanos , Masculino , Pessoa de Meia-Idade , Qualidade da Assistência à Saúde/organização & administração , Qualidade da Assistência à Saúde/tendências , Encaminhamento e Consulta/organização & administração , Encaminhamento e Consulta/tendências , Reino Unido , Serviços Urbanos de Saúde/organização & administraçãoRESUMO
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.
Assuntos
Artrite Reumatoide/psicologia , Cultura , Avaliação da Deficiência , Comportamento de Doença , Adaptação Psicológica , Idoso , Estudos Transversais , Feminino , Humanos , Controle Interno-Externo , Masculino , Pessoa de Meia-Idade , Qualidade de Vida/psicologia , Inquéritos e QuestionáriosRESUMO
PURPOSE: To investigate whether people with long term conditions, whatever their specific nature, need to be assessed and treated for the full range of mental, physical and social problems. Main question investigated: that rheumatoid arthritis and schizophrenia will be associated with significantly greater impairment across the subscores of the SF36 scale than in reference general population samples. Specific hypothesis tested: while rheumatoid arthritis and schizophrenia will impair both physical and mental functioning, when comparing the two groups there will be a greater difference between the physical component scores than there will be between the mental/emotional component scores of the short form health survey (SF-36). METHODS: Cross sectional comparison of SF-36 subscore profiles of cohorts of: (1) people with rheumatoid arthritis attending specialist Rheumatology outpatient clinics in five London hospitals (n = 446), and (2) people with schizophrenia treated by community psychiatric teams in four sites in Europe (n = 409). RESULTS: Both groups had greater impairments across the whole spectrum of mental and physical problems assessed by the SF-36 than age specific normative data for the general population. The results also support our hypothesis that, comparing the people with rheumatoid arthritis and schizophrenia, we did find that there is a greater discrepancy between the physical scales than there is between the mental/emotional scales of the SF-36. CONCLUSIONS: These findings show that whether the primary long-term condition is presenting as physical or as mental disorder, the practitioner should ensure that the full range of physical, mental and social problems is assessed and treated.