Non-viral chimeric antigen receptor (CAR) T cells going viral.

Chimeric antigen receptor (CAR) T cell therapy has made significant strides in the treatment of B-cell malignancies, but its application in treating solid tumors still poses significant challenges. Particularly, the widespread use of viral vectors to deliver CAR transgenes into T cells comes with limitations, including high costs and regulatory restrictions, which hinder the translation of novel genetic engineering concepts into clinical applications. Non-viral methods, such as transposon/transposase and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, offer promising alternatives for stable transgene insertion in CAR-T cells. These methods offer the potential to increase accessibility and efficiency in the development and delivery of CAR-T cell therapies. The main challenge in using non-viral methods, however, is their low knock-in efficiency, which leads to low transgene expression levels. In this review, we discuss recent developments in non-viral approaches for CAR-T cell production, the manufacturing requirements for clinical-grade production of non-viral CAR-T cells, and the adjustments needed in quality control for proper characterization of genomic features and evaluation of potential genotoxicity.

Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

An empowerment model of workplace support following disclosure, for people with MS.

BACKGROUND: Vocational interventions aimed at increasing job retention for people with multiple sclerosis (MS) are reliant upon a partnership with a supportive work environment. A better understanding of the types of psychosocial support that are most conducive to retaining employees' sense of work-efficacy will enhance the success of interventions aimed at reducing workplace barriers to job maintenance. OBJECTIVE: The objective of this study is to identify the types of psychosocial support that people with MS require post-disclosure, in order to maintain their employment status. In particular, we examined the roles of psychological safety and work-efficacy. METHODS: We interviewed 40 employees with MS either individually (n = 25) or within three focus groups (n = 15). These interviews were audio-taped and the content analysed, using an inductive thematic approach. RESULTS: Themes to emerge in organisational responses to disclosure were: a focus on ability (leading to enhanced perceptions of psychological safety and higher work-efficacy) and on disability (leading to diminished psychological safety and reduced perceptions of work-efficacy). CONCLUSION: Organisational responses to disclosure demonstrating trust and inclusive decision making, and focussing on employee abilities, enhance perceptions of psychological safety at work. This increases the likelihood that employees with MS will retain their sense of work-efficacy and reduce their intentions to leave.

Disclosure of diagnosis of multiple sclerosis in the workplace positively affects employment status and job tenure.

BACKGROUND: For many employees with multiple sclerosis (MS), disclosure of their diagnosis at work is seen as a high-risk strategy that might lead to diminished perceptions of their capabilities by supervisors and colleagues, if not outright discrimination. The consequence of this mistrust surrounding the disclosure process is that employees with MS may leave it until too late to effectively manage symptoms at work. OBJECTIVE: The objective of this paper is to statistically evaluate the relationship between disclosure of diagnosis at work and maintenance of employment. METHODS: Three annual, large-sample self-report surveys of MS patients prospectively examined the relationship between disclosure of diagnosis at work and employment status. A total of 1438 people responded to all three surveys. Of employed persons in 2010 (n = 946), 673 also responded to the 2012 survey. Of these 673 respondents 564 were still employed. RESULTS: People who had disclosed their MS status to an employer were more likely to remain in employment in Year 3. The effect of disclosure in predicting employment status remained after controlling for age, gender, hours worked and level of disability. CONCLUSION: This study provides the first empirical support for the positive role of disclosure in maintaining employment status, measured both as job retention and tenure in current employment.

Exercise therapy and recovery after SCI: evidence that shows early intervention improves recovery of function.

STUDY DESIGN: This was designed as an experimental study. OBJECTIVES: Locomotor training is one of the most effective strategies currently available for facilitating recovery of function after an incomplete spinal cord injury (SCI). However, there is still controversy regarding the timing of treatment initiation for maximal recovery benefits. To address this issue, the present study compares the effects of exercise initiated in the acute and secondary phase of SCI. SETTING: Texas A&M University, College Station, TX, USA. METHODS: Rats received a moderate spinal contusion injury and began an exercise program 1 (D1-EX) or 8 days (D8-EX) later. They were individually placed into transparent exercise balls for 60 min per day, for 14 consecutive days. Control rats were placed in exercise balls that were rendered immobile. Motor and sensory recovery was assessed for 28 days after injury. RESULTS: The D1-EX rats recovered significantly more locomotor function (BBB scale) than controls and D8-EX rats. Moreover, analyses revealed that rats in the D8-EX group had significantly lower tactile reactivity thresholds compared with control and D1-EX rats, and symptoms of allodynia were not reversed by exercise. Rats in the D8-EX group also had significantly larger areas of damage across spinal sections caudal to the injury center compared with the D1-EX group. CONCLUSION: These results indicate that implementing an exercise regimen in the acute phase of SCI maximizes the potential for recovery of function.

The effect of group-based exercise on cognitive performance and mood in seniors residing in intermediate care and self-care retirement facilities: a randomised controlled trial.

OBJECTIVE: To determine the effect of a general group-based exercise programme on cognitive performance and mood among seniors without dementia living in retirement villages. DESIGN: Randomised controlled trial. SETTING: Four intermediate care and four self-care retirement village sites in Sydney, Australia. PARTICIPANTS: 154 seniors (19 men, 135 women; age range 62 to 95 years), who were residents of intermediate care and self-care retirement facilities. INTERVENTION: Participants were randomised to one of three experimental groups: (1) a general group-based exercise (GE) programme composed of resistance training and balance training exercises; (2) a flexibility exercise and relaxation technique (FR) programme; or (3) no-exercise control (NEC). The intervention groups (GE and FR) participated in 1-hour exercise classes twice a week for a total period of 6 months. MAIN OUTCOME MEASURES: Using standard neuropsychological tests, we assessed cognitive performance at baseline and at 6-month re-test in three domains: (1) fluid intelligence; (2) visual, verbal and working memory; and (3) executive functioning. We also assessed mood using the Geriatric Depression Scale (GDS) and the Positive and Negative Affect Schedule (PANAS). RESULTS: The GE programme significantly improved cognitive performance of fluid intelligence compared with FR or NEC. There were also significant improvements in the positive PANAS scale within both the GE and FR groups and an indication that the two exercise programmes reduced depression in those with initially high GDS scores. CONCLUSIONS: Our GE programme significantly improved cognitive performance of fluid intelligence in seniors residing in retirement villages compared with our FR programme and the NEC group. Furthermore, both group-based exercise programmes were beneficial for certain aspects of mood within the 6-month intervention period.

An explanation for the apparent dissociation between clinical remission and continued structural deterioration in rheumatoid arthritis.

OBJECTIVE: Achieving remission is the aim of treatment in rheumatoid arthritis (RA). This should represent minimal arthritis activity and ensure optimal disease outcome. However, we have previously demonstrated a high prevalence of imaging-detected synovial inflammation in RA patients who were in clinical remission. The purpose of this study was to evaluate the long-term significance of subclinical synovitis and its relationship to structural outcome. METHODS: We studied 102 RA patients receiving conventional treatment who had been judged by their consultant rheumatologist to be in remission, as well as 17 normal control subjects. Subjects underwent clinical, laboratory, functional, and quality of life assessments over 12 months. In addition to standard radiography of the hands and feet, imaging of the hands and wrists was performed with musculoskeletal ultrasonography (US) and conventional 1.5 T magnetic resonance imaging (MRI) at baseline and 12 months, using validated acquisition and scoring techniques. RESULTS: Despite their being in clinical remission, 19% of the patients displayed deterioration in radiographic joint damage over the study period. Scores on musculoskeletal US synovial hypertrophy, power Doppler (PD), and MRI synovitis assessments in individual joints at baseline were significantly associated with progressive radiographic damage (P=0.032, P<0.001, and P=0.002, respectively). Furthermore, there was a significant association between the musculoskeletal US PD score at baseline and structural progression over 12 months in totally asymptomatic metacarpophalangeal joints (P=0.004) and 12 times higher odds of deterioration in joints with increased PD signal (odds ratio 12.21, P<0.001). CONCLUSION: Subclinical joint inflammation detected by imaging techniques explains the structural deterioration in RA patients in clinical remission who are receiving conventional therapy. Our findings reinforce the utility of imaging for the accurate evaluation of disease status and the prediction of structural outcome.

Abnormal T cell differentiation persists in patients with rheumatoid arthritis in clinical remission and predicts relapse.

OBJECTIVES: An abnormal CD4+ T cell subset related to inflammation exposure (inflammation-related cells, IRC) has been identified in rheumatoid arthritis (RA). Patients with inflammatory and non-inflammatory diseases were used to examine the relationship between inflammation and this T cell subset in vivo. METHODS: Blood was collected from healthy controls and patients with RA (active disease or in clinical remission), Crohn's disease and osteoarthritis. IRC and chemokine receptors were quantified by flow cytometry. Thymic activity and apoptotic factors were measured by real-time polymerase chain reaction. Circulating cytokines were measured by enzyme-linked immunosorbent assay. CXCR4 and SDF1 in synovial biopsies were measured using immunohistochemistry. RESULTS: IRC were identified in patients with RA (p<0.0001) and Crohn's disease (p = 0.005), but not in those with osteoarthritis. In RA in remission, IRC persisted (p<0.001). In remission, hyperproliferation of IRC was lost, chemokine receptor expression was significantly lowered (p<0.007), Bax expression dropped significantly (p<0.001) and was inversely correlated with IRC (rho = -0.755, p = 0.03). High IRC frequency in remission was associated with relapse within 18 months (OR = 6.4, p<0.001) and a regression model predicted 72% of relapse. CONCLUSIONS: These results suggest a model in which, despite the lack of systemic inflammation, IRC persist in remission, indicating that IRC are an acquired feature of RA. They have, however, lost their hyper-responsiveness, acquired a potential for survival, and no longer express chemokine receptors. IRC persistence in remission confirms their important role in chronic inflammation as circulating precursors of pathogenic cells. This was further demonstrated by much higher incidence of relapse in patients with high IRC frequency in remission.

Lower uterine segment involvement as a predictor for lymph node spread in endometrial carcinoma.

OBJECTIVES: Several predictive factors for lymph node spread in endometrial cancer have been identified including tumor grade, depth of invasion, lymphatic or vascular-space invasion, and histologic subtype. Lower uterine segment involvement may also be predictive of lymph node spread. The objective of this study was to investigate the relationship between lower uterine segment involvement in endometrial carcinoma and lymph node spread. METHODS: This was an IRB approved retrospective study. Data were collected for all patients diagnosed with endometrial cancer from June 1999 to December 2004. The primary end point was the presence of nodal involvement. Subset analysis was performed by histologic subtype. Univariate and multivariate nominal logistic regression was performed. Categorical variables were compared using Chi-square and Fischer's Exact Test. RESULTS: Two-hundred and ninety-nine subjects were eligible for review. One-hundred seventy four (58%) had lower uterine segment involvement. Forty-four (25%) of those with lower uterine segment involvement had positive nodes compared to 10 (8%) of those without (p=0.0001). On univariate analysis, lower uterine segment involvement, lymphovascular-space invasion, and deep invasion predicted nodal disease. On multivariate analysis, lower uterine segment remained predictive of nodal spread for the endometrioid subset. For high-risk histologies, only lymphovascular-space invasion and deep myometrial invasion were predictive of nodal spread. CONCLUSIONS: Lower uterine segment involvement in endometrial carcinoma is an important predictor of lymph node involvement for patients with endometrioid histologies. Tumor within the lower uterine segment may be an important factor to consider in intraoperative decision making regarding staging.

The development of an evidence-based educational framework to facilitate the training of competent rheumatologist ultrasonographers.

OBJECTIVES: Musculoskeletal ultrasonography (MSKUS) has been described by some rheumatologists as the 'stethoscope of the joint'. Such enthusiasm is supported by evidence confirming validity and clinical utility in evaluation of musculoskeletal diseases. But if rheumatologist-performed MSKUS is to emulate the impact of cardiologist-performed echocardiography, a number of educational challenges need be addressed. Evaluation of current training reveals the absence of a unified educational structure, ad hoc teaching and assessment and published data are insufficient to make practice and training recommendations specific to rheumatology. METHODS: Informed by developments in adult-learning theory, we have utilized a competency-based approach to develop an educational programme for rheumatologist ultrasonographers. Fundamental to this process has been accurate functional analysis of the role of the rheumatologist ultrasonographer and precise translation of these data into educational outcomes. This involved a thorough, transparent, iterative, curriculum-defining approach, employing quantitative and qualitative research techniques including interview, questionnaire, Delphi and standard setting methodologies. All relevant stakeholders were engaged, including international US experts and clinical rheumatologists. RESULTS: Outcomes include clarification of the role of a rheumatologist ultrasonographer; definition of appropriate knowledge and skills; establishment of competency standards; a balanced, clearly defined, clinically relevant educational outcome blueprint. Teaching and assessment approaches have been piloted as part of an accessible modular curriculum strategy. Thorough validation and evaluation confirms effectiveness, efficiency and suitability. CONCLUSIONS: A comprehensive evidence-based, expert consensus-defined educational framework is proposed that provides a template for teaching and learning and standards for competency assessment. This should facilitate common principles of training, uniform professional practice and a justifiable governance structure.

The challenges of integrating ultrasonography into routine rheumatology practice: addressing the needs of clinical rheumatologists.

OBJECTIVES: The practice of musculoskeletal ultrasonography (MSKUS) by UK rheumatologists remains limited, despite their reported enthusiasm. This study aimed to investigate factors that may encourage or limit future dissemination of rheumatologist-performed MSKUS and provide insights into perceived clinical importance and learning motivation relating to published recommendations by MSKUS experts. METHODS: A written questionnaire study was conducted, involving 48 rheumatologists. Questions included the potential role of self-performed MSKUS, skills that they would be willing to learn and factors that may encourage or limit learning and practice. Competency recommendations proposed by imaging experts (142 skills in 7 anatomical areas) were reviewed, and quantitative and qualitative data collected regarding 'value to their practice' and 'learning motivation'. RESULTS: Eighty-nine percent wished to learn MSKUS. Factors influencing learning and practice included time to achieve competency; relative-added clinical value of MSKUS examination; limited training infrastructure; access to existing imaging service; equipment funding. Skills offering greatest clinical utility were inflammatory arthritis assessment and guided procedures; least useful were evaluation of ligament/muscle lesions and soft tissue masses. There was a close correlation between clinical utility, learning motivation and competency standard. CONCLUSIONS: A trade-off between added clinical value and time to achieve competency is the major factor influencing practice and training in MSKUS. Most rheumatologists report limited time to devote to training and therefore need to prioritize areas of importance for dedicated learning. Educational programmes need to be highly focused and relevant to clinical and job-plan requirements in order to encourage future dissemination of MSKUS practice by rheumatologists.

The prognostic significance of lower uterine segment involvement in surgically staged endometrial cancer patients with negative nodes.

OBJECTIVES: Endometrial cancer is the most common female genital malignancy in the United States. Stage is the most important prognostic factor. Other factors include grade, lymph-vascular space invasion (LVI), and myometrial invasion. Tumor location in the lower uterine segment (LUS) may also be important. LUS involvement correlates with nodal involvement, and nodal involvement is an important prognostic indicator. This study investigates the importance of LUS involvement in patients with pathologically negative nodes. METHODS: This was an IRB approved retrospective study. Data were collected for patients diagnosed with endometrial cancer from June 1999 to September 2004. Patients who underwent nodal evaluation with no evidence of nodal disease were eligible for analysis. The primary endpoint was progression-free survival. Secondary endpoints included recurrence rate and overall survival. Analysis was performed with the JMP5.1 statistical program. RESULTS: 285 patients were identified. 85 were excluded because they received postoperative care elsewhere. 3 charts were missing, and 15 pathology reports did not mention LUS. 147 of the remaining 182 subjects had negative nodes and formed the study population. 57% of these subjects had LUS involvement. Follow-up was similar for those with and without LUS involvement at 74 vs. 73 months respectively. PFS was similar at 70 and 63 months in those with and without LUS involvement (p=0.2). Recurrence correlated with LUS involvement on univariate analysis, however, not on multivariate analysis. CONCLUSIONS: In endometrial cancer patients with negative nodes, disease within the lower uterine segment does not imply a worse prognosis. The previously described implications of LUS involvement are likely due to the strong association of LUS disease with lymph node spread.

Presence of significant synovitis in rheumatoid arthritis patients with disease-modifying antirheumatic drug-induced clinical remission: evidence from an imaging study may explain structural progression.

OBJECTIVE: More timely and effective therapy for rheumatoid arthritis (RA) has contributed to increasing rates of clinical remission. However, progression of structural damage may still occur in patients who have satisfied remission criteria, which suggests that there is ongoing disease activity. This questions the validity of current methods of assessing remission in RA. The purpose of this study was to test the hypothesis that modern joint imaging improves the accuracy of remission measurement in RA. METHODS: We studied 107 RA patients receiving disease-modifying antirheumatic drug therapy who were judged by their consultant rheumatologist to be in remission and 17 normal control subjects. Patients underwent clinical, laboratory, functional, and quality of life assessments. The Disease Activity Score 28-joint assessment and the American College of Rheumatology remission criteria, together with strict clinical definitions of remission, were applied. Imaging of the hands and wrists using standardized acquisition and scoring techniques with conventional 1.5T magnetic resonance imaging (MRI) and ultrasonography (US) were performed. RESULTS: Irrespective of which clinical criteria were applied to determine remission, the majority of patients continued to have evidence of active inflammation, as shown by findings on the imaging assessments. Even in asymptomatic patients with clinically normal joints, MRI showed that 96% had synovitis and 46% had bone marrow edema, and US showed that 73% had gray-scale synovial hypertrophy and 43% had increased power Doppler signal. Only mild synovial thickening was seen in 3 of the control subjects (18%), but no bone marrow edema. CONCLUSION: Most RA patients who satisfied the remission criteria with normal findings on clinical and laboratory studies had imaging-detected synovitis. This subclinical inflammation may explain the observed discrepancy between disease activity and outcome in RA. Imaging assessment may be necessary for the accurate evaluation of disease status and, in particular, for the definition of true remission.

Early rheumatoid arthritis is associated with a deficit in the CD4+CD25high regulatory T cell population in peripheral blood.

OBJECTIVE: Our aim was to test the hypothesis that there is a deficit in the CD4+CD25high regulatory T-cell population in early rheumatoid arthritis (RA), either in size or functional activity. METHODS: Peripheral blood mononuclear cells were examined from subjects with early active RA who had received no previous disease-modifying therapy (n = 43), from individuals with self-limiting reactive arthritis (n = 14), from subjects with stable, well-controlled RA (n = 82) and from healthy controls (n = 72). The frequencies of CD4+CD25high T-cells were quantified using flow cytometry, and function was assessed by the ability to suppress proliferation of CD4+CD25- T-cells. Paired blood and synovial fluid was analysed from a small number of RA and reactive arthritis patients. RESULTS: There was a smaller proportion of CD4+CD25high T-cells in the peripheral blood of early active RA patients (mean 4.25%) than in patients with reactive arthritis or in controls (mean 5.90 and 5.30%, respectively, P = 0.001 in each case). Frequencies in stable, well-controlled RA (mean 4.63%) were not significantly different from early active RA or controls. There were no differences in suppressor function between groups. Higher frequencies of CD4+CD25high T-cells were found in synovial fluid than blood in both RA and reactive arthritis. CONCLUSIONS: These data demonstrate a smaller CD4+CD25high regulatory T-cell population in peripheral blood of individuals with early active RA prior to disease-modifying treatment. This may be a contributory factor in the susceptibility to RA and suggests novel approaches to therapy.

Ultrasonography for rheumatologists: the development of specific competency based educational outcomes.

BACKGROUND: A competency based approach to the education of rheumatologists in musculoskeletal ultrasonography (MSK US) ensures standards are documented, transparent, accountable, and defensible, with clear benefit to all stakeholders. Specific competency outcomes will facilitate informed development of a common curriculum and structured programme of training and assessment. OBJECTIVE: To determine explicit competency based learning outcomes for rheumatologists undertaking MSK US. METHODS: International experts in MSK US, satisfying specific selection criteria, were asked to define the minimum standards required by a rheumatologist to be judged competent in MSK US. They reviewed 115 MSK US skills, comprising bone and soft tissue pathology, in seven joints regions of the upper and lower limbs, and rated their relative importance according to specific criteria. These data are presented as specific educational outcomes within designated competency categories. RESULTS: 57 expert MSK US practitioners were identified and 35 took part in this study. Ten generic core competency outcomes were recognised including physics, anatomy, technique, and interpretation. Regarding specific regional competencies, 53% (61/115) were considered "must know" core learning outcomes, largely comprising inflammatory joint/tendon/bone pathology and guided procedures; 45% (52/115) were required at an intermediate/advanced level (18/115 "should know", 34/115 "could know"), and 2% (2/115) were deemed inappropriate/unnecessary for rheumatologist ultrasonographers. CONCLUSIONS: This is the first study to developing a competency model for the education of rheumatologists in MSK US based on the evidence of international experts. A specific set of learning outcomes has been defined, which will facilitate future informed education and practice development and provide a blueprint for a structured rheumatology MSK US curriculum and assessment process.

Abnormal cervical cytology: a risk factor for endometrial cancer recurrence.

The objective of this study was to evaluate the relationship between cervical cytology, histologic type, and risk of endometrial cancer recurrence. We performed a retrospective study of patients undergoing surgery for endometrial carcinoma. Risk factors for recurrence including histology, tumor grade, nodal status, myometrial invasion, peritoneal washings, stage, and cervical cytology were assessed. Abnormal cervical cytology was defined as the presence of any endometrial cells on Pap smear. Papillary serous and clear cell carcinomas were considered high-risk histologies. Univariate and multivariate analyses of risk factors for recurrence were performed. Thirty-nine (9%) patients developed recurrent endometrial cancer. More patients with abnormal Pap smears recurred (12% versus 4%, P < 0.05). For endometrioid adenocarcinoma, abnormal cervical cytology occurred in 61% and 7% recurred, while with high-risk histologies, 84% had abnormal cervical cytology and 19% recurred (P < 0.05). Other significant predictors of recurrence on univariate analysis were myometrial invasion, nodal status, washings, stage, and histology. On multivariate analysis, only nodal status remained a significant predictor of recurrence. Abnormal cervical cytology is associated with increased risk of endometrial cancer recurrence. Abnormal cervical cytology occurs more frequently in high-risk histologies, which are known to have a higher risk of recurrence. On multivariate analysis, only nodal spread remains a significant predictor of recurrence.