Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of cell cycle.

Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols reliant on culture. However, the kinetics and mechanisms by which this occurs remain incompletely characterized. Here, through time-resolved scRNA-Seq, matched in vivo functional analysis and the use of a reversible in vitro system of early G1 arrest, we define the sequence of transcriptional and functional events occurring during the first ex vivo division of human LT-HSCs. We demonstrate that the sharpest loss of LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limiting global variability in gene expression and transiently upregulating gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programmes in culture. However, contrary to current assumptions, we demonstrate that loss of HSC function ex vivo is independent of cell cycle progression. Finally, we show that targeting LT-HSC adaptation to culture by inhibiting early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrates that controlling early LT-HSC adaptation to ex vivo culture, for example via JAK inhibition, is of critical importance to improve HSC gene therapy and expansion protocols.

Identification of a small molecule for enhancing lentiviral transduction of T cells.

Genetic modification of cells using viral vectors has shown huge therapeutic benefit in multiple diseases. However, inefficient transduction contributes to the high cost of these therapies. Several transduction-enhancing small molecules have previously been identified; however, some may be toxic to the cells or patient, otherwise alter cellular characteristics, or further increase manufacturing complexity. In this study, we aimed to identify molecules capable of enhancing lentiviral transduction of T cells from available small-molecule libraries. We conducted a high-throughput flow-cytometry-based screen of 27,892 compounds, which subsequently was narrowed down to six transduction-enhancing small molecules for further testing with two therapeutic lentiviral vectors used to manufacture GSK's clinical T cell therapy products. We demonstrate enhanced transduction without a negative impact on other product attributes. Furthermore, we present results of transcriptomic analysis, suggesting alteration of ribosome biogenesis, resulting in reduced interferon response, as a potential mechanism of action for the transduction-enhancing activity of the lead compound. Finally, we demonstrate the ability of the lead transduction enhancer to produce a comparable T cell product using a 3-fold reduction in vector volume in our clinical manufacturing process, resulting in a predicted 15% reduction in the overall cost of goods.

Development of an automated manufacturing process for large-scale production of autologous T cell therapies.

Engineered T cell therapies have shown significant clinical success. However, current manufacturing capabilities present a challenge in bringing these therapies to patients. Furthermore, the cost of development and manufacturing is still extremely high due to complexity of the manufacturing process. Increased automation can improve quality and reproducibility while reducing costs through minimizing hands-on operator time, allowing parallel manufacture of multiple products, and reducing the complexity of technology transfer. In this article, we describe the results of a strategic alliance between GSK and Miltenyi Biotec to develop a closed, automated manufacturing process using the CliniMACS Prodigy for autologous T cell therapy products that can deliver a high number of cells suitable for treating solid tumor indications and compatible with cryopreserved apheresis and drug product. We demonstrate the ability of the T cell Transduction - Large Scale process to deliver a significantly higher cell number than the existing process, achieving 1.5 × 1010 cells after 12 days of expansion, without affecting other product attributes. We demonstrate successful technology transfer of this robust process into three manufacturing facilities.

Antifungal therapies for allergic bronchopulmonary aspergillosis in people with cystic fibrosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is an allergic reaction to colonisation of the lungs with the fungus Aspergillus fumigatus, and affects around 10% of people with cystic fibrosis. ABPA is associated with an accelerated decline in lung function. High doses of corticosteroids are the main treatment for ABPA; although the long-term benefits are not clear, and their many side effects are well-documented. A group of compounds, the azoles, have activity against A fumigatus, and have been proposed as an alternative treatment for ABPA. Of this group, itraconazole is the most active. A separate antifungal compound, amphotericin B, has been used in aerosolised form to treat invasive infection with A fumigatus, and may have potential for the treatment of ABPA. Antifungal therapy for ABPA in cystic fibrosis needs to be evaluated. This is an update of a previously published review. OBJECTIVES: The review aimed to test the hypotheses that antifungal interventions for the treatment of ABPA in cystic fibrosis: 1. improve clinical status compared to placebo or standard therapy (no placebo); and 2. do not have unacceptable adverse effects. If benefit was demonstrated, we planned to assess the optimal type, duration, and dose of antifungal therapy. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register, which comprises references identified from comprehensive electronic database searches, handsearches of relevant journals, and abstract books of conference proceedings. Date of the most recent search of the Group's Trials Register was 28 September 2021. We searched ongoing trials registries, most recently on 11 March 2022. Earlier, we also approached pharmaceutical companies regarding possible unpublished trials. SELECTION CRITERIA: Published or unpublished randomised controlled trials, in which antifungal treatments were compared to either placebo or no treatment, or where different doses of the same treatment were used in the treatment of ABPA in people with cystic fibrosis. DATA COLLECTION AND ANALYSIS: The searches identified six trials; none of which met the inclusion criteria for the review. MAIN RESULTS: We included no completed randomised controlled trials. There is currently one ongoing trial, which we may find eligible for a future update. AUTHORS' CONCLUSIONS: At present, there are no randomised controlled trials that evaluate the use of antifungal therapies for the treatment of ABPA in people with cystic fibrosis, although one trial is currently ongoing. Trials with clear outcome measures are needed to properly evaluate the use of corticosteroids in people with ABPA and cystic fibrosis.

Recommendations for procurement of starting materials by apheresis for advanced therapy medicinal products.

Activities involved in the production of certain advanced therapy medicinal products (ATMPs) require standardized approaches to mononuclear cell procurement to ensure the highest product quality, safety and process efficiency. These aims must be achieved while meeting regulatory and accreditation requirements for the procurement of mononuclear cells as starting materials. Mononuclear cells constitute the starting materials for many ATMPs, and this article sets out recommendations for procurement by clinical apheresis, addressing the variation among existing working practices and different manufacturers' requirements that currently poses a challenge when managing multiple different protocols.

Analysis of Community-Acquired Urinary Tract Infection Treatment in Pediatric Patients Requiring Hospitalization: Opportunity for Use of Narrower Spectrum Antibiotics.

Background: The most narrow-spectrum antibiotic possible should be used for empiric and definitive treatment of pediatric urinary tract infections (UTIs). Objectives: The objectives of this study were to determine an appropriate narrow-spectrum antibiotic for empiric UTI treatment, factors differentiating empiric first-generation cephalosporin (FGC) versus third-generation cephalosporin (TGC) coverage, and factors associated with unnecessarily broad-spectrum definitive antibiotic treatment. Methods: This was a retrospective chart review of children admitted from 2013 to 2015 who were diagnosed with a UTI and received treatment. Multivariable logistic regression assessed independent factors associated with our outcomes. Results: Of 568 diagnosed UTIs, 88.6% received empiric TGC treatment. Empiric coverage among cultured organisms was only 5.4% lower in FGC versus TGC. Adolescent age group (odds ratio [OR] = 8.83, 95% confidence interval [CI] = 1.47-53.11), uncircumcised males (OR = 4.52, 95% CI = 1.27-16.08), Hispanic ethnicity (OR = 4.37, 95% CI = 1.14-16.82), and hospitalization within the preceding 3 months (OR = 4.73, 95% CI = 1.38-16.23) were associated with FGC nonsusceptibility among TGC susceptible Enterobacteriaceae pathogens. De-escalation occurred in 55.8% of diagnosed UTIs eligible for de-escalation at discharge. Urine white blood cell (WBC) count >5 (OR = 2.89, 95% CI = 1.14-7.21), serum WBC count (OR = 1.04, 95% CI = 1.01-1.07), and having only one narrow-spectrum treatment option (OR = 5.1, 95% CI = 2.43-10.66) were associated with unnecessarily broad-spectrum definitive treatment. Conclusion and Relevance: FGC would be an appropriate narrow-spectrum empiric agent for UTIs at our institution. The factors associated with FGC nonsusceptibility can further stratify empiric treatment decisions. The factors associated with unnecessarily broad-spectrum definitive treatment illustrate areas for educational efforts and future research regarding UTI treatment.

Changes in microRNA expression during differentiation of embryonic and induced pluripotent stem cells to definitive endoderm.

Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to treat type 1 diabetes through cell replacement therapy. However, the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification, and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation, and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified, suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR, and the relationship between one of these miRNAs, miR-151a-5p, and its predicted target gene, SOX17, was investigated by luciferase assay, and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion, these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells.

The 49th hour: analysis of a follow-up medication and vaccine dispensing field test.

Anthrax response plans, exercises, and assessments over the past decade have focused almost exclusively on the first 48 hours of the public health response following a jurisdiction-wide exposure and provision of an initial 10-day supply of antibiotics from the Strategic National Stockpile (SNS). But mass dispensing of the subsequent 50-day course of antibiotics and administration of the 3-dose vaccine series have received considerably less attention, although these follow-up activities may prove even more complex. In 2014, the Philadelphia Department of Public Health (PDPH) made its first foray into this next frontier of mass prophylaxis planning by: (1) researching patient safety and adherence considerations relevant to the antibiotics in the SNS; (2) designing a model for a second-visit antibiotic and vaccine point of dispensing (POD), including development of an enhanced screening protocol that assumes a higher level of medical responsibility; and (3) field testing this model during a real seasonal influenza vaccination clinic to assess throughput and accuracy and to evaluate the resources needed to operationalize this model. While the observations and data presented here provide some framework for local long-term mass prophylaxis planning efforts, many areas remain undefined, including the distribution of responsibilities among the public health and healthcare communities to ensure patient safety. In addition to presenting findings, the larger intent of this article is to initiate a dialogue with other stakeholders at the local, state, and federal levels to advance long-term mass prophylaxis planning.

The role of microRNAs in the pancreatic differentiation of pluripotent stem cells.

The generation of ß-cells in vitro is an attractive option for cell therapy treatments for type 1 diabetes and also for the development of more accurate disease models. A number of studies have demonstrated that insulin-expressing cells can be generated by the in vitro differentiation of human pluripotent stem cells. However, to date, these differentiation protocols are often inefficient, time-consuming and highly variable. In many cases, this is a result of an incomplete understanding of the regulatory processes involved in the differentiation of human pluripotent stem cells. One such process is the control of gene expression by microRNAs (miRNAs). Given that miRNAs have the potential to influence cell fate, we present in this short review the evidence that a further understanding of the role of miRNAs in pancreatic development and function may be important in the on-going quest to generate insulin-secreting cells from pluripotent stem cells.

Effectiveness of a neonatal intensive care unit access intercom linked audiovisual display monitor highlighting infection control procedures.

This prospective interventional study aimed at increasing knowledge and adherence to 4 infection control standards by visitors to a neonatal intensive care unit. Visitors were interviewed and observed for knowledge of and adherence to the standards pre- and postinstallation of an audiovisual display monitor, which demonstrates handwashing and delivers an auditory and written list of the standards. Handwashing adherence and watch removal improved from 79.2% to 100% and 67% to 89.7%, respectively. Recall of the standards increased from 19% to 81%.