Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

The PapilloCheck Assay for Detection of High-Grade Cervical Intraepithelial Neoplasia.

Human papillomavirus (HPV) testing is used in primary cervical screening, as an adjunct to cervical cytology for the management of low grade abnormal cytology, and in a test of cure. PapilloCheck (Greiner Bio-One) is a PCR-based DNA microarray system that can individually identify 24 HPV types, including the 13 high-risk (HR) types identified by Hybrid Capture 2 (HC2). Here, we compare PapilloCheck with HC2 for the detection of high-grade cervical intraepithelial neoplasia (CIN2+) in a total of 8,610 cervical cytology samples from the ARTISTIC population-based cervical screening study. We performed a retrospective analysis of 3,518 cytology samples from round 1 ARTISTIC enriched for underlying CIN2+ (n = 723) and a prospective analysis of 5,092 samples from round 3 ARTISTIC. Discrepant results were tested using the Roche reverse line blot (RLB) or Linear Array (LA) assay. The relative sensitivity and specificity of HR PapilloCheck compared with that of HC2 for the detection of CIN2+ in women aged over 30 years were 0.94 (95% confidence interval [CI], 0.91, 0.97) and 1.05 (95% CI, 1.04, 1.05), respectively. HC2 missed 44/672 (7%) CIN2+ lesions, while HR PapilloCheck missed 74/672 (11%) CIN2+ lesions. Thirty-six percent of HC2-positive normal cytology samples were HR HPV negative by both PapilloCheck and RLB/LA, indicating that the use of HR PapilloCheck rather than HC2 in population-based primary screening would reduce the number of additional tests required (e.g., reflex cytology) in women where underlying CIN2+ is extremely unlikely. HR PapilloCheck could be a suitable HPV detection assay for use in the cervical screening setting.

Influence of resuspension volume on dry sampling devices taken for human papillomavirus testing: implications for self-sampling.

Optimization of technical parameters that influence the performance of human papillomavirus (HPV) testing on self-taken samples is important. Here, the authors assessed the impact of resuspension volume on the detection of HPV using four validated HPV assays. Two self-sampling devices, FLOQSwabs® and Evalyn® Brushes, were inoculated with dilutions of HPV-16-positive cell line, then resuspended in various volumes of ThinPrep. The influence of vortexing during resuspension was also assessed. At target concentrations around the assay cutoff, larger volumes led to decreased HPV detection. Interestingly, the effect(s) of vortexing differed by the self-sampling device. Resuspension in 5 ml or less may maximize the detection of HPV sequences. Using a proxy of clinical material, the current observations underline the importance of optimizing preanalytical laboratory processes to support high-quality HPV testing of self-samples.

Two self-sampling strategies for HPV primary cervical cancer screening compared with clinician-collected sampling: an economic evaluation.

OBJECTIVE: To compare the costs and effects of three sampling strategies for human papillomavirus (HPV) primary screening. DESIGN: Cost-consequence analysis from a health system perspective using a deterministic decision tree model. SETTING: England. PARTICIPANTS: A cohort of 10 000 women aged 25-65 years eligible for the National Health Service Cervical Screening Programme (NHSCSP). METHODS: The model was based on the NHSCSP HPV primary screening pathway and adapted for self-sampling. It used a 3-year cycle: routine screening (year 1) and recall screening (years 2/3). Parameter inputs were informed using published studies, NHSCSP reports and input from experts and manufacturers. Costs were from 2020/2021, British pound sterling (£). INTERVENTIONS: Three sampling strategies were implemented: (1) routine clinician-collected cervical sample, (2) self-collected first-void (FV) urine, (3) self-collected vaginal swab. The hypothetical self-sampling strategies involved mailing women a sampling kit. MAIN OUTCOME MEASURES: Primary outcomes: overall costs (for all screening steps to colposcopy), number of complete screens and cost per complete screen. SECONDARY OUTCOMES: number of women screened, number of women lost to follow-up, cost per colposcopy and total screening costs for a plausible range of uptake scenarios. RESULTS: In the base case, the average cost per complete screen was £56.81 for clinician-collected cervical sampling, £38.57 for FV urine self-sampling and £40.37 for vaginal self-sampling. In deterministic sensitivity analysis, the variables most affecting the average cost per screen were the cost of sample collection for clinician-collected sampling and the cost of laboratory HPV testing for the self-sampling strategies. Scaled to consider routine screening in England, if uptake in non-attenders increased by 15% and 50% of current screeners converted to self-sampling, the NHSCSP would save £19.2 million (FV urine) or £16.5 million (vaginal) per year. CONCLUSION: Self-sampling could provide a less costly alternative to clinician-collected sampling for routine HPV primary screening and offers opportunities to expand the reach of cervical screening to under-screened women.

Changes to culture pH and dissolved oxygen can enhance chimeric antigen receptor T-cell generation and differentiation.

Chimeric antigen receptor (CAR) T-cell therapy is growing clinically and commercially as a powerful new approach to treat cancer. Understanding how key culture conditions such as pH and dissolved oxygen (DO) affect CAR T-cell generation and function is important in developing better CAR-T manufacturing processes and CAR T-cell therapies for patients. We used the automated mini-bioreactor (AMBR) 15 platform to assess how differences in pH and DO affect CAR T-cell transduction, proliferation, and differentiation. We found that higher pH can significantly improve CAR T-cell transduction and proliferation, and also biases CAR T-cells away from an effector memory and toward a more central memory phenotype. Both high and low DO negatively affect CAR T-cell generation, with both hypoxic and hyperoxic conditions reducing T-cell transduction into CAR T-cells. Collectively, this data underscores how pH and DO can significantly affect CAR T-cell expansion and differentiation, and provides insight into the optimal culture conditions to enhance CAR T-cell yield and phenotype in clinical and commercial processes.

Enabling Allogeneic T Cell-Based Therapies: Scalable Stirred-Tank Bioreactor Mediated Manufacturing.

Allogeneic T cells are key immune therapeutic cells to fight cancer and other clinical indications. High T cell dose per patient and increasing patient numbers result in clinical demand for a large number of allogeneic T cells. This necessitates a manufacturing platform that can be scaled up while retaining cell quality. Here we present a closed and scalable platform for T cell manufacturing to meet clinical demand. Upstream manufacturing steps of T cell activation and expansion are done in-vessel, in a stirred-tank bioreactor. T cell selection, which is necessary for CAR-T-based therapy, is done in the bioreactor itself, thus maintaining optimal culture conditions through the selection step. Platform's attributes of automation and performing the steps of T cell activation, expansion, and selection in-vessel, greatly contribute to enhancing process control, cell quality, and to the reduction of manual labor and contamination risk. In addition, the viability of integrating a closed, automated, downstream process of cell concentration, is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while preserving key factors of cell quality and process control.

Cross-sectional study of HPV testing in self-sampled urine and comparison with matched vaginal and cervical samples in women attending colposcopy for the management of abnormal cervical screening.

OBJECTIVES: Human papillomavirus (HPV) testing in cervical screening offers the potential for self-sampling to improve uptake among non-attenders. High-risk (HR) HPV detection in urine shows promise, but few studies have examined its sensitivity for cervical intraepithelial neoplasia (CIN2+) detection compared with standard cervical samples. The aims of this cross-sectional study were to optimise conditions for urine testing for HPV detection; to determine concordance for HR-HPV detection in matched urine, vaginal and cervical samples; to compare the sensitivity of HR-HPV testing for the detection of CIN2+ in matched samples; and to determine the acceptability of urine testing for cervical screening. DESIGN: Cross-sectional study. SETTING: Secondary care colposcopy clinic in North West England. PARTICIPANTS: Women aged 25 years of age or older, attending colposcopy clinic for management of abnormal cervical screening results or a suspicious-looking cervix. In total, 104 women took part in the study. Triple matched samples were available for 79 and 66 women using Abbott RealTime (ART) and Roche Cobas 4800 (RC), respectively. INTERVENTION: Self-collected urine and vaginal samples and practitioner-obtained cervical samples were tested for HR-HPV by ART and RC assays, including comparison of neat and preservative-fixed urine. Colposcopic opinion was recorded and directed cervical biopsies taken if clinically indicated. The acceptability of self-testing was evaluated by questionnaire. PRIMARY OUTCOME MEASURE: The sensitivity of urine to detect underlying CIN2+. SECONDARY OUTCOME MEASURES: The comparative sensitivity of vaginal and cervical samples to detect CIN2+; the acceptability of urine sampling. RESULTS: Preservative-fixed, but not neat urine, showed good concordance with vaginal samples for the detection of HR-HPV. The sensitivity for detecting CIN2+ was 15/18 (83%) for urine and 16/18 (89%) for cervical and vaginal samples by ART, and 15/17 (88%) for all samples by RC. Urine-based testing was broadly acceptable to women. CONCLUSIONS: Urinary HR-HPV detection offers an alternative strategy of cervical screening. Larger studies to determine its clinical utility are warranted.

Transcriptional Profiling of Mesenchymal Stem Cells Identifies Distinct Neuroimmune Pathways Altered by CNS Disease.

BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stem cells (BM-MSCs) are an attractive cell based therapy in the treatment of CNS demyelinating diseases such as multiple sclerosis (MS). Preclinical studies demonstrate that BM-MSCs can effectively reduce clinical burden and enhance recovery in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS. However, a number of recent clinical trials have not shown significant functional benefit following BM-MSC infusion into MS patients. One possibility for the discrepancy between animal and human studies is the source of the cells, as recent studies suggest BM-MSCs from MS patients or animals with EAE lack reparative efficacy compared to naïve cells. We sought to define important transcriptional and functional differences between diseased and naïve MSCs. METHODS AND RESULTS: We utilized RNA Sequencing (RNA-Seq) to assess changes in gene expression between BM-MSCs derived from EAE animals and those derived from healthy controls. We show that EAE alters the expression of a large number of genes in BM-MSCs and changes in gene expression are more pronounced in chronic versus acute disease. Bioinformatic analysis revealed extensive perturbations in BM-MSCs in pathways related to inflammation and the regulation of neural cell development. These changes suggest that signals from EAE derived BM-MSCs inhibit rather than enhance remyelination, and in-vitro studies showed that conditioned medium from EAE MSCs fails to support the development of mature oligodendrocytes, the myelinating cells of the CNS. CONCLUSIONS: These data provide insight into the failure of autologous BM-MSCs to promote recovery in MS and support the concept of utilizing non-autologous MSCs in future clinical trials.

CNS disease diminishes the therapeutic functionality of bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have emerged as a potentially powerful cellular therapy for autoimmune diseases including multiple sclerosis (MS). Based on their success in treating animal models of MS like experimental autoimmune encephalomyelitis (EAE), MSCs have moved rapidly into clinical trials for MS. The majority of these trials use autologous MSCs derived from MS patients, although it remains unclear how CNS disease may affect these cells. Here, we report that bone marrow MSCs derived from EAE mice lack therapeutic efficacy compared to naïve MSCs in their ability to ameliorate EAE. Treatment with conditioned medium from EAE-MSCs also fails to modulate EAE, and EAE-MSCs secrete higher levels of many pro-inflammatory cytokines compared to naïve MSCs. Similarly, MSCs derived from MS patients have less therapeutic efficacy than naïve MSCs in treating EAE and secrete higher levels of some of the same pro-inflammatory cytokines. Thus diseases like EAE and MS diminish the therapeutic functionality of bone marrow MSCs, prompting reevaluation about the ongoing use of autologous MSCs as a treatment for MS.

MSC Therapeutics in Chronic Inflammation.

The utilization of mesenchymal stem cells (also known as mesenchymal stromal cells, or MSCs) as a cell-based therapy for diseases that have ongoing inflammatory damage has become increasingly available. Our understanding of the cell biology of MSCs is still incomplete. However, as a result of increasing numbers of pre-clinical and clinical studies, general themes are emerging. The capacity of MSCs to reduce disease burden is largely associated with their ability to modulate the activity of the host immune responses rather than to contribute directly to tissue regeneration. As a result, they have significant potential in the treatment of chronic inflammatory disease regardless of the affected tissue. For example, MSC based therapies have been developed in the context of diseases as diverse as rheumatoid arthritis and multiple sclerosis. Here we discuss some of the principles that link these conditions, and the aspects of MSC biology that contribute to their use as a therapy for chronic inflammatory conditions.

A prospective study of anal cancer screening in HIV-positive and negative MSM.

OBJECTIVE: The study sought to establish the feasibility and acceptability of anal screening among men MSM. DESIGN: Prospective cohort study. SETTING: Sexual health clinics in tertiary care. PATIENTS: Known HIV-positive and negative MSM who have anoreceptive intercourse. INTERVENTION: Anal screening with human papilloma virus (HPV) testing, liquid-based cytology and high-resolution anoscopy with biopsy of anoscopic abnormalities. Participants completed questionnaires at baseline and at 6 months. RESULTS: Anal HPV was highly prevalent in MSM (HIV-positive, 88% and HIV-negative, 78%). Despite the high prevalence of cytological abnormality in both HIV-positive (46.2%) and negative (35.0%) MSM, almost half of anal intraepithelial neoplasia (AIN) of all grades were associated with negative cytology. Anoscopically directed biopsies detected AIN3 or worse (AIN3+) in 14 of 203 (6.9%) of HIV-positive MSM and three of 81 (3.7%) HIV-negative MSM. The corresponding prevalence of AIN2+ was 26.6 and 20.9%, respectively. One case of AIN3 was detected at the second visit. Screening was considered to be highly acceptable by participants. CONCLUSION: The high prevalence of high-risk-HPV and frequency of false negative cytology in this study suggest that high-resolution anoscopy would have most clinical utility, as a primary screening tool for anal cancer in a high-risk group. The prevalence of AIN3+ in HIV-positive MSM lends support for a policy of screening this group, but the high prevalence of lower grade lesions which do not warrant immediate treatment and the limitations of treating high-grade lesions requires careful consideration in terms of a screening policy.

Type-specific HPV prevalence in invasive cervical cancer in the UK prior to national HPV immunisation programme: baseline for monitoring the effects of immunisation.

AIMS: To establish the human papillomavirus (HPV) type-specific prevalence in cervical cancer and high-grade cervical lesions in the UK prior to the introduction of national HPV vaccination. METHODS: Specimens of cervical cancer (n=1235) and cervical intraepithelial neoplasia (CIN)3 (n=2268) were tested for HPV genotypes in England, Scotland, Wales and Northern Ireland. Data were pooled and weighted estimates presented. RESULTS: Among cervical cancer cases, 95.8% were positive for at least one high-risk (HR) HPV type. Restricting to those with HR HPV, the proportion positive for HPV16 and/or HPV18 was similar across countries (weighted overall prevalence 83.0%). This proportion decreased with increasing age at diagnosis (p=0.0005). HPV31, HPV33, HPV45, HPV52 and/or HPV58 were detected in 16.1% of HR HPV-positive cervical cancers and there was no significant association with age for these types. For HR HPV-positive CIN3 cases, there was a similar age-specific pattern with the highest positivity of HPV16 and/or HPV18 in the youngest age group (77.2%). The proportion of HR HPV CIN3 cases positive for HPV31, HPV33, HPV45, HPV52 and/or HPV58 was 36.3% in those aged <30 years at diagnosis. CONCLUSIONS: The prevalence of HPV 16 and/or 18 was high in all UK countries and highest in those diagnosed at a younger age. The UK is well placed to monitor the impact of HPV vaccination on type-specific HPV prevalence in cervical disease.