Comparability of automated human induced pluripotent stem cell culture: a pilot study.

Consistent and robust manufacturing is essential for the translation of cell therapies, and the utilisation automation throughout the manufacturing process may allow for improvements in quality control, scalability, reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently, the effects of manual centrifugation and automated non-centrifugation process steps, performed using TAP Biosystems' CompacT SelecT automated cell culture platform, upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study, has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However, non-centrifugation hiPSC populations exhibited greater cell yields, greater aggregate rates, increased pluripotency marker expression, and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures, and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories.

Manufacturing models permitting roll out/scale out of clinically led autologous cell therapies: regulatory and scientific challenges for comparability.

Manufacturing of more-than-minimally manipulated autologous cell therapies presents a number of unique challenges driven by complex supply logistics and the need to scale out production to multiple manufacturing sites or near the patient within hospital settings. The existing regulatory structure in Europe and the United States imposes a requirement to establish and maintain comparability between sites. Under a single market authorization, this is likely to become an unsurmountable burden beyond two or three sites. Unless alternative manufacturing approaches can be found to bridge the regulatory challenge of comparability, realizing a sustainable and investable business model for affordable autologous cell therapy supply is likely to be extremely demanding. Without a proactive approach by the regulators to close this "translational gap," these products may not progress down the development pipeline, threatening patient accessibility to an increasing number of clinician-led autologous cellular therapies that are already demonstrating patient benefits. We propose three prospective manufacturing models for the scale out/roll out of more-than-minimally manipulated clinically led autologous cell therapy products and test their prospects for addressing the challenge of product comparability with a selected expert reference panel of US and UK thought leaders. This paper presents the perspectives and insights of the panel and identifies where operational, technological and scientific improvements should be prioritized. The main purpose of this report is to solicit feedback and seek input from key stakeholders active in the field of autologous cell therapy in establishing a consensus-based manufacturing approach that may permit the roll out of clinically led autologous cell therapies.

Reduced overtriage and undertriage with a new triage system in an urban accident and emergency department in Botswana: a cohort study.

BACKGROUND: Improvements in triage have demonstrated improved clinical outcomes in resource-limited settings. In 2009, the Accident and Emergency (A&E) Department at the Princess Marina Hospital (PMH) in Botswana identified the need for a more objective triage system and adapted the South African Triage Scale to create the PMH A&E Triage Scale (PATS). AIM: The primary purpose was to compare the undertriage and overtriage rates in the PATS and pre-PATS study periods. METHODS: Data were collected from 5 April 2010 to 1 May 2011 for the PATS and compared with a database of patients triaged from 1 October 2009 to 24 March 2010 for the pre-PATS. Data included patient disposition outcomes, demographics and triage level assignments. RESULTS: 14 706 (pre-PATS) and 25 243 (PATS) patient visits were reviewed. Overall, overtriage rates improved from 53% (pre-PATS) to 38% (PATS) (p<0.001); likewise, undertriage rates improved from 47% (pre-PATS) to 16% (PATS) (p<0.001). Statistically significant decreases in both rates were found when paediatric and adult cases were analysed separately. PATS was more predictive of inpatient admission, Intensive Care Unit (ICU) admission and death rates in the A&E than was the pre-PATS. The lowest acuity category of each system had a 0.6% (pre-PATS) and 0% (PATS) chance of death in the A&E or ICU admission (p<0.001). No change in death rate was seen between the pre-PATS and PATS, but ICU admission rates decreased from 0.35% to 0.06% (p<0.001). CONCLUSIONS: PATS is a more predictive triage system than pre-PATS as evidenced by improved overtriage, undertriage and patient severity predictability across triage levels.

Cerebrotendinous Xanthomatosis, a Treatable Disorder Often Missed: Case Series of Three Patients Confirmed by Genetic Testing.

ABSTRACT: Cerebrotendinous xanthomatosis (CTX) is a treatable autosomal recessive disorder with varied clinical manifestations and age of onset and is often diagnosed late. We report three cases of CTX who presented at our center with clinical features of frequent diarrhea, early cataracts, xanthomas, cognitive decline, ataxia, neuropathy, and other manifestations of CTX. Magnetic resonance imaging (MRI) brain in all three patients revealed abnormalities consistent with CTX. Diagnosis was confirmed by next-generation sequencing. Chenodeoxycholic acid (CDCA) is recommended as the drug of choice, as it can halt the disease progression and reverse some of the symptoms. In addition to late diagnosis, nonavailability of CDCA in our part of world adds to the problem of management of such patients; therefore, they are often started on alternative therapies, which are less effective.

An unusual case of refractory metabolic acidosis.

Homeopathy is one of the most frequently used systems of complementary and alternative medicine. The patients who seek homeopathic treatment are primarily those suffering from long-standing, chronic diseases. These medicines may have considerable risk of severe side effects. Some homeopathic medicinal preparations use alcohol as a base and are frequently prescribed for common conditions. We hereby report an unusual case of refractory metabolic acidosis after homeopathic medicinal treatment.

Isolated neurobrucellosis-characteristic clinical and laboratory features.

To report various neurological syndromes, CSF findings, imaging and diagnostic methods used in neurobrucellosis patients admitted in our Neurology department over a period of 6 years. Case records of patients admitted to our department from August 2014 to May 2020 were searched for neurobrucellosis and data were obtained. A total of 19 patients were diagnosed as neurobrucellosis over a period of 6 years. Ten patients had chronic meningitis, five had VIII nerve involvement, one had optic neuritis, two had acute meningitis, one had subacute meningitis, four had myelopathy, five had polyradiculitis and two had spondylodiscitis. CSF was abnormal in 17 patients. Neutrophilic pleocytosis was seen in 12 patients who included nine patients with chronic symptomatology. Brain imaging was abnormal in three chronic meningitis patients. One had diffuse meningeal enhancement, another had hydrocephalus while the third patient had meningeal enhancement with basal exudates and contrast enhancement of bilateral VIII nerve. One of the patients of acute meningitis had hydrocephalus while the other one had bilateral T2/FLAIR hyperintensities with enhancement of meninges and leptomeningeal vessels. Elevated antibody titers only in serum was seen in six patients while elevated antibody titers only in CSF was seen in seven patients. Four patients had elevated antibody titers in both serum and CSF. CSF culture was positive in three patients. Neurobrucellosis is a rare clinical complication of brucellosis but may pose a problem in diagnosis as it can mimic tuberculosis. Involvement of VIII nerve and neurophilic pleocytosis in CSF despite chronic symptomatology can be diagnostic clues favoring neurobrucellosis.

Miller Fisher syndrome associated with COVID 19.

Miller Fisher syndrome (MFS), is an acute peripheral neuropathy, a variant of Guillain-Barre syndrome, that develops following exposure to different viral, bacterial, and fungal pathogens. Patients usually present with a triad of ophthalmoplegia, ataxia, and areflexia. During Covid pandemic MFS has been described associated with novel coronavirus disease 2019 (COVID-19). Here we describe the clinical course, Cerebrospinal fluid (CSF) findings, nerve conduction studies, treatment and outcome of the patient having MFS concurrent with COVID 19.

MicroRNA-128 inhibits mitochondrial biogenesis and function via targeting PGC1α and NDUFS4.

The size and morphology of mitochondria are very heterogeneous and correlates well with their healthy functioning. In many pathological conditions, mitochondrial morphology is altered due to impaired mitochondrial dynamics (a collective term for mitochondrial fusion and fission) and dysfunction. The current study aimed at identifying the role of microRNA-128 (miR-128) in regulating mitochondrial biogenesis. Previously, peroxisome proliferator activator receptor γ coactivator 1α (PGC1α) has been shown to co-activate key intermediates of mitochondrial biogenesis, function, and dynamics; however, the upstream regulatory network remains largely unknown. We, herein using in silico analysis followed by in vitro experiments in C2C12 myoblasts, showed that miR-128 reduces mitochondrial biogenesis by directly targeting PGC1α. The expression of downstream genes, nuclear respiratory factors 1 and 2 (NRF1 and NRF2, respectively), and mitochondrial transcription factor A (TFAM) were decreased in C2C12 myoblasts upon overexpression of miR-128. Also, miR-128 is shown to promote mitochondrial dysfunction by directly targeting NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (NDUFS4). The mitochondrial dynamics and morphology were impaired post miR-128 overexpression, as revealed by downregulation of fusion proteins (mitofusin1 and 2, i.e., MFN1 and MFN2, respectively) and upregulation of fission protein (dynamin-related protein 1, i.e., DRP1). Conversely, inhibition of miR-128 expression improved mitochondrial biogenesis, function, and dynamics, as evidenced by increased mitochondrial mass and ATP production after antimiR-128 treatment. Our findings reveal that inhibition of miR-128 can be a new potential target for reversing the effects of metabolic disorders of skeletal muscle as observed during many pathophysiological conditions such as obesity and type II diabetes.

A meta-analysis of comorbidities in COVID-19: Which diseases increase the susceptibility of SARS-CoV-2 infection?

Comorbidities in COVID-19 patients often lead to more severe outcomes. The disease-specific molecular events, which may induce susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, are being investigated. To assess this, we retrieved array-based gene expression datasets from patients of 30 frequently occurring acute, chronic, or infectious diseases. Comparative analyses of the datasets were performed after quantile normalization and log2 transformation. Among the 78 host genes prominently implicated in COVID-19 infection, ACE2 (receptor for SARS-CoV-2) was positively regulated in several cases, namely, leukemia, psoriasis, lung cancer, non-alcoholic fatty liver disease (NAFLD), breast cancer, and pulmonary arterial hypertension (PAH). FURIN was positively regulated in some cases, such as leukemia, psoriasis, NAFLD, lung cancer, and type II diabetes (T2D), while TMPRSS2 was positively regulated in only 3 cases, namely, leukemia, lung cancer, and T2D. Genes encoding various interferons, cytokines, chemokines, and mediators of JAK-STAT pathway were positively regulated in leukemia, NAFLD, and T2D cases. Among the 161 genes that are positively regulated in the lungs of COVID-19 patients, 99-111 genes in leukemia (including various studied subtypes), 77 genes in NAFLD, and 48 genes in psoriasis were also positively regulated. Because of the high similarity in gene expression patterns, the patients of leukemia, NAFLD, T2D, psoriasis, and PAH may need additional preventive care against acquiring SARS-CoV-2 infections. Further, two genes CARBONIC ANHYDRASE 11 (CA11) and CLUSTERIN (CLU) were positively regulated in the lungs of patients infected with either SARS-CoV-2, or SARS-CoV or Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

Inhibition of microRNA-128-3p attenuates hypercholesterolemia in mouse model.

AIMS: Hypercholesterolemia remains a critical risk factor for cardiovascular diseases and there is an urgent need to develop effective alternative therapeutics. Herein, we investigated the effects of miR-128-3p inhibition on serum cholesterol levels using a hypercholesterolemic mouse model. MATERIALS AND METHODS: Five injections of anti-miR-128-3p (AM-128) treatment were given, and the cholesterol profile in serum and liver was quantified. We validated the underlying gene network using qRT-PCR, western blotting, ELISA, and dual luciferase assays. KEY FINDINGS: AM-128 treatment inhibits cholesterol biosynthesis by upregulating INSIG1 and downregulating HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) expression. The serum cholesterol clearance by SR-B1 (scavenger receptor class B member 1) and LDLR (low density lipoprotein receptors) was also increased. Furthermore, the catabolism of cholesterol by CYP7A1 (cytochrome P450 family 7 subfamily A member 1) was increased. SIGNIFICANCE: Our results confirmed a critical role of miR-128-3p inhibition in lowering serum cholesterol and suggest its potential therapeutic implications in reversing hypercholesterolemia.

Distributed automated manufacturing of pluripotent stem cell products.

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site-decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing 'live' corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.

Automated, scalable culture of human embryonic stem cells in feeder-free conditions.

Large-scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor-intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder-free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1-81, and SSEA-4), stable karyotype, and multi-germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell-use in clinical, scientific, and industrial applications.

The management of risk and investment in cell therapy process development: a case study for neurodegenerative disease.

Cell-based therapies must achieve clinical efficacy and safety with reproducible and cost-effective manufacturing. This study addresses process development issues using the exemplar of a human pluripotent stem cell-based dopaminergic neuron cell therapy product. Early identification and correction of risks to product safety and the manufacturing process reduces the expensive and time-consuming bridging studies later in development. A New Product Introduction map was used to determine the developmental requirements specific to the product. Systematic Risk Analysis is exemplified here. Expected current value-based prioritization guides decisions about the sequence of process studies and whether and if an early abandonment of further research is appropriate. The application of the three tools enabled prioritization of the development studies.

Advancing research on the economic value of emergency care.

Emergency care and the emergency care system encompass an array of time-sensitive interventions to address acute illness and injury. Research has begun to clarify the enormous economic burden of acute disease, particularly in low-income and middle-income countries, but little is known about the cost-effectiveness of emergency care interventions and the performance of health financing mechanisms to protect populations against catastrophic health expenditures. We summarise existing knowledge on the economic value of emergency care in low resource settings, including interventions indicated to be highly cost-effective, linkages between emergency care financing and universal health coverage, and priority areas for future research.

Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, biomanufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells.

Tuberculosis among individuals with community-acquired pneumonia presenting to emergency in Gaborone, Botswana.

Delays in diagnosing Tuberculosis (TB) are associated with increased transmission. TB may present as a clinical syndrome that mimics community-acquired pneumonia (CAP). The aim of this paper was to determine frequency of TB among patients with CAP at a referral hospital in Gaborone, Botswana. We performed a retrospective study of adults presenting with CAP from April 2010-October 2011 to the Emergency Department (ED); we matched this cohort to the National Botswana Tuberculosis Registry (NBTR) to identify individuals subsequently diagnosed with TB. We assessed demographics, time to TB diagnosis, clinical outcomes and performed logistic regressions to identify factors associated with TB diagnosis. We identified 1305 individuals presenting with CAP; TB was subsequently diagnosed in 68 (5.2%). The median time to TB diagnosis was 9.5 days. Forty percent were AFB sputum smear positive and 87% were identified as being HIV-positive. Subsequent diagnosis of TB is common among individuals with CAP at our ED, suggesting that TB may be present at the time of CAP presentation. Given the lack of distinguishing clinical factors between pulmonary TB and CAP, adults presenting with CAP should be evaluated for active TB in Botswana.

Quality control guidelines for clinical-grade human induced pluripotent stem cell lines.

Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for the generation of cellular therapeutics requires demonstration of comparability of lines derived from different individuals and in different facilities. This requires agreement on the critical quality attributes of such lines and the assays that should be used. Working from established recommendations and guidance from the International Stem Cell Banking Initiative for human embryonic stem cell banking, and concentrating on those issues more relevant to iPSCs, a series of consensus workshops has made initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade, which are outlined in this report. Continued evolution of this field will likely lead to revision of these guidelines on a regular basis.

Science-based assessment of source materials for cell-based medicines: report of a stakeholders workshop.

Human pluripotent stem cells (hPSCs) have the potential to transform medicine. However, hurdles remain to ensure safety for such cellular products. Science-based understanding of the requirements for source materials is required as are appropriate materials. Leaders in hPSC biology, clinical translation, biomanufacturing and regulatory issues were brought together to define requirements for source materials for the production of hPSC-derived therapies and to identify other key issues for the safety of cell therapy products. While the focus of this meeting was on hPSC-derived cell therapies, many of the issues are generic to all cell-based medicines. The intent of this report is to summarize the key issues discussed and record the consensus reached on each of these by the expert delegates.

Control of pore size and structure of tissue engineering scaffolds produced by supercritical fluid processing.

Tissue engineering scaffolds require a controlled pore size and structure to host tissue formation. Supercritical carbon dioxide (scCO2) processing may be used to form foamed scaffolds in which the escape of CO2 from a plasticized polymer melt generates gas bubbles that shape the developing pores. The process of forming these scaffolds involves a simultaneous change in phase in the CO2 and the polymer, resulting in rapid expansion of a surface area and changes in polymer rheological properties. Hence, the process is difficult to control with respect to the desired final pore size and structure. In this paper, we describe a detailed study of the effect of polymer chemical composition, molecular weight and processing parameters on final scaffold characteristics. The study focuses on poly(DL-lactic acid) (PDLLA) and poly(DL-lactic acid-co-glycolic acid) (PLGA) as polymer classes with potential application as controlled release scaffolds for growth factor delivery. Processing parameters under investigation were temperature (from 5 to 55 degrees C) and pressure (from 60 to 230 bar). A series of amorphous PDLLA and PLGA polymers with various molecular weights (from 13 KD to 96 KD) and/or chemical compositions (the mole percentage of glycolic acid in the polymers was 0, 15, 25, 35 and 50 respectively) were employed. The resulting scaffolds were characterised by optical microscopy, scanning electron microscopy (SEM), and micro X-ray computed tomography (microCT). This is the first detailed study on using these series polymers for scaffold formation by supercritical technique. This study has demonstrated that the pore size and structure of the supercritical PDLLA and PLGA scaffolds can be tailored by careful control of processing conditions.