Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long‐term culture of HSPCs. In this study, a novel three‐dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34⁺CD38^?) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long‐term culture of primitive HSPCs in a microfluidic environment.     

Evaluation of an ovary‐on‐a‐chip in large mammalian models: Species specificity and influence of follicle isolation status

The ability to grow oocytes from immature ovarian follicles in vitro has significant potential for fertility preservation; yet, it has proved challenging in large mammalian species due to the complex metabolic needs and long‐term culture requirements. Currently, follicular incubations are based on a “static” system with manual exchange of medium. Despite the numerous advantages of conventional culturing approaches, recapitulating the native microenvironment and supporting the survival of ovarian follicles from large mammalian species still represent challenges. In this study, we utilized an innovative, dynamic microfluidic system to support the in vitro survival of domestic cat and dog follicles enclosed within the ovarian cortex or isolated from ovarian cortex. Results indicate both species‐specific and tissue type‐specific differences in response to microfluidic culture. Domestic cat but not dog ovarian cortical tissues maintained viability under flow similar to conventional agarose gel controls. Preantral stage isolated follicles from both species that grew most favourably in conventional alginate bead culture, but overall, there was no influence of culture system on expression of follicle development or oocyte health markers. This system represents an important exploration toward the development of an improved ovarian in vitro culture system of large mammalian species (e.g., cats and dogs), which has potential applications for fertility preservation, reproductive toxicology, and endangered mammal conservation efforts.     

Kidney‐in‐a‐lymph node: A novel organogenesis assay to model human renal development and test nephron progenitor cell fates

Stem cell‐derived organoids are emerging as sophisticated models for studying development and disease and as potential sources for developing organ substitutes. Unfortunately, although organoids containing renal structures have been generated from mouse and human pluripotent stem cells, there are still critical unanswered questions that are difficult to attain via in vitro systems, including whether these nonvascularized organoids have a stable and physiologically relevant phenotype or whether a suitable transplantation site for long‐term in vivo studies can be identified. Even orthotopic engraftment of organoid cultures in the adult does not provide an environment conducive to vascularization and functional differentiation. Previously, we showed that the lymph node offers an alternative transplantation site where mouse metanephroi can differentiate into mature renal structures with excretory, homeostatic, and endocrine functions. Here, we show that the lymph node lends itself well as a niche to also grow human primary kidney rudiments and can additionally be viewed as a platform to interrogate emerging renal organoid cultures. Our study has a wide‐ranging impact for tissue engineering approaches to rebuild functional tissues in vivo including—but not limited to—the kidney.     

Implementation of a diabetes clinic‐in‐a‐clinic project in a family practice setting: using the plan,do, study,act model

Objectives. The American Association of Diabetic Educators suggests that educating patients about their diabetes management facilitates problem solving and coping skills. This paper will describe a clinic‐in‐a‐clinic model of care delivery founded on the principles of the Chronic Care Model and focused towards the outcomes proposed by the American Association of Diabetic Educators. The reader will be introduced to the use of the ‘plan, do, study, act’ process used to develop this model in a clinical setting. Background. Self‐management support, a key component of the Chronic Care Model, focuses on providing patients with the skills to make healthcare decisions. Self‐management encourages patient to be responsible for his/her own health care. Because diabetes outcomes and complication prevalence are related to the degree of self involvement in illness care, self‐management support is an important component of disease management. Design. Plan, do, study, act model for program development. Methods. The ‘plan, do, study, act’ cycles outlined the steps needed to implement the clinic‐in‐a‐clinic program with success related to coordination of all components and continual assessment and revision. Each cycle was initiated in a sequential order allowing for evaluation and goal adjustment before the next cycle was implemented. Conclusions. The majority of patients seen were middle‐aged, obese, females with HbA1cs greatly above the recommended 7·0. Patients selected a variety of topics related to diabetes management for their clinical session. Participation rates were consistent with regular clinic visit attendance. Barriers to success of the program were related to both structure and process. Relevance to clinical practice. The clinic‐in‐a‐clinic design moves disease management from individual practice into a property of the health systems and places importance on the collaboration of patient, provider and delivery system in reducing the consequences of chronic illness. Use of the ‘plan, do, study, act’ cycle model offers a method for changing the process of care delivery in a structured, sequential approach.     

Comparative analysis of marketed factor VIII products: recombinant products are not alike vis‐a‐vis soluble protein aggregates and subvisible particles

Essentials

• Aggregation is a critical quality attribute of protein therapeutics influencing immunogenicity.
• Aggregates and subvisible particles in 9 recombinant factor VIII (rFVIII) products were analyzed.
• Major differences in aggregate and particle concentrations were detected after reconstitution.
• rFVIII product quality determined aggregation propensity under use‐relevant stress.

Summary

Background

Recombinant protein technologies have facilitated the development of novel factor VIII (FVIII) therapeutics with improved production efficiency, potency and half‐live, and a low risk of viral transmission. The increasing number of recombinant FVIII (rFVIII) products and information on their efficacy, safety and cost allow patients and healthcare professionals to adjust treatment to individual needs. Nonetheless, 20–32% of previously untreated patients with severe hemophilia A develop inhibitory antibodies to rFVIII following treatment. The root cause of the immunogenicity of rFVIII products is not well understood. Data for human interferon and human insulin products suggest that critical quality parameters such as soluble protein aggregates (SPAs) and subvisible particles (SVPs) influence the immunogenicity of protein therapeutics. Therefore, we analyzed SPA and SVP concentrations in commercially available rFVIII products and determined how these parameters change upon exposure of rFVIII products to relevant stress conditions.

Objectives

Compare critical quality parameters such as SPA and SVP concentrations in rFVIII products under intended use and use‐relevant stress conditions.

Methods

Nine rFVIII products (≥ 3 lots each) were analyzed by high‐performance liquid chromatography‐size exclusion chromatography (HPLC‐SEC) and flow cytometry‐based particle analysis.

Results/conclusions

SPAs and SVPs were present at different concentrations in all freshly reconstituted rFVIII products: SPA concentrations ranged from 0.2% to 11.6%; SVPs were 0.7 × 10⁶ to 114.0 × 10⁶ / 1000 IU. Under use‐relevant stress conditions (agitation and shear stress) the products formed additional SPAs and SVPs to different degrees. The collected data indicate that product quality determines its propensity to form SVPs and SPAs, and highlights differences between marketed rFVIII products.     

Once‐a‐week psychiatric ward round or daily inpatient team meeting? A multidisciplinary mental health team's experience of new ways of working

Evidence indicates that while service users are dissatisfied with current ward round practices, studies of how professionals experience this practice are sparse. This study highlights staff view of the once‐a‐week psychiatric ward round compared to a reformed ward round taking place every weekday. Interviews were conducted at one acute psychiatric ward in north‐west England. Our analysis revealed a core theme, ‘forming a new way of working’, which could be understood from three themes. The theme, ‘bound by tradition’, emphasized how the traditional ward round represented a double‐edged sword: it provided a safe structure, but it also highlighted a shared awareness of an urgent need to leave old ways of working behind. The process of change became discernable in the themes ‘juggling the change’ and ‘light at the end of the tunnel’, which showed that restructuring the traditional ward round was both possible and valued. We found that staff views on ward rounds are more complex than had been earlier understood, but new ways of working can be implemented, if the impact of tradition, the process of change, and the time to bed down are taken into account.     

Upregulation of miR-23a∼27a∼24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a~27a~24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 3′UTR based reporter construct. Moreover, we also showed that over expression of miR-23a~27a~24-2 sensitized HEK293T cells to TNF-α cytotoxicity. Taken together, our study demonstrates that enhanced TNF-α induced apoptosis in HEK293T cells by over expression of miR-23a~27a~24-2 cluster provides new insights in the development of novel therapeutics for cancer.     

LINC00240 knockdown inhibits nasopharyngeal carcinoma progress by targeting miR‐26a‐5p

ObjectiveThis study intended to explore the regulatory functions of LINC00240 on nasopharyngeal carcinoma (NPC).MethodsMiR‐26a‐5p inhibitor, mimic, and siLINC00240 were transfected into NPC cells. QRT‐PCR was employed to assess miR‐26a‐5p and LINC00240 expressions. The targeting relationship of LINC00240 and miR‐26a‐5p was analyzed through dual luciferase reporter and RNA immunoprecipitation assay. Cell counting kit‐8 assay, colony formation assay, flow cytometry assay, wound healing assay, Transwell assay and in vitro angiogenesis assay were adopted for the evaluation of the effects of LINC00240 or miR‐26a‐5p and LINC00240 on NPC cells regarding cell proliferation, apoptosis and cycle, migration, invasion, and angiogenesis. EZH2, cell cycle, and epithelial‐mesenchymal transition (EMT)‐related protein expression was tested through Western blot.ResultsLINC00240 had a high expression in NPC tissues and cell lines. Silenced LINC00240 significantly suppressed the 5‐8F and HK1 cell proliferation, invasion, migration, and angiogenesis, but raised cell apoptosis, and cells were blocked in G0/G1 phase. MiR‐26a‐5p was a target of LINC00240. MiR‐26a‐5p upregulation suppressed the NPC cell proliferation, migration, invasion, angiogenesis, N‐cadherin and EZH2 expression, while it elevated apoptosis and p21, p27 and E‐cadherin expressions, whereas miR‐26a‐5p downregulation performed conversely. LINC00240 knockdown partially offset the effects of miR‐26a‐5p downregulation on cell proliferation, migration, invasion, angiogenesis, apoptosis, and EZH2.ConclusionLINC00240 knockdown restrained cell proliferation, invasion, migration, and angiogenesis, while it advanced apoptosis via miR‐26a‐5p in NPC by EZH2 inhibition.     

Danish general practitioners’ management of patients with COPD: a nationwide survey

Background In Denmark, general practitioners (GPs) have the main responsibility for chronic obstructive pulmonary disease (COPD) management. Internationally, COPD appears to be significantly under-treated, which could be explained by ‘therapeutic nihilism’ or lack of knowledge.Aim To investigate: (1) To what extent COPD management provided by GPs includes the core elements of pharmacological treatment, smoking cessation and physical activity, and (2) To what extent GPs need educational support and consulting with a specialist in pulmonary medicine.Design A national cross-sectional web-based survey conducted in April–June 2019. The survey included items on COPD management and educational support needs.Setting Danish general practice.Subjects A population of approximately 3400 GPs (all GPs in Denmark).Results We received response from 470 GPs (14% response rate). Overall, the respondents reported that they offered COPD management including all relevant treatment elements. Smoking cessation was supported in 58% and physical activity was supported in 23% of the respondents. Future consultations on smoking cessation were planned by 35% and physical activity by 15% respondents. GPs responded to ‘needing educational support in COPD management’ to a ‘high degree’ in 8% and to ‘some degree’ in 43%.Conclusion The survey suggested that COPD maintenance support provided by GPs seemed to be inadequate regarding smoking cessation and physical activity. Moreover, some GPs expressed a need for educational support in COPD management. More research is needed to understand the potential barriers to evidence-based delivery of COPD-management.

Key points

• In Denmark, general practitioners (GPs) have the main responsibility for the management of chronic obstructive pulmonary disease (COPD).
• The present study shows that non-pharmacological interventions such as supporting smoking cessation and particularly promoting physical activity received less attention than pharmacological treatment.
• The study suggests a need for educational support of the GPs in COPD management.