Investigating NF-κB signaling in lung fibroblasts in 2D and 3D culture systems.

BACKGROUND: Inflammatory respiratory diseases are amongst major global health challenges. Lung fibroblasts have been shown to play a key role in lung inflammatory responses. However, their exact role in initiation and maintenance of lung diseases has remained elusive partly due to the limited availability of physiologically relevant in vitro models. Therefore, developing new tools that enable investigating the molecular pathways (e.g. nuclear factor-kappa B (NF-κB) activation) that underpin inflammatory responses in fibroblasts could be a valuable resource for scientists working in this area of research. RESULTS: In order to investigate NF-κB activation in response to pro-inflammatory stimuli in real-time, we first developed two detection systems based on nuclear localization of NF-κB by immunostaining and luciferase reporter assay system. Furthermore using electrospun porous scaffolds, with similar geometry to human lung extracellular matrix, we developed 3D cultures of lung fibroblasts allowing comparing NF-κB activation in response to pro-inflammatory stimuli (i.e. TNF-α) in 2D and 3D. Our data clearly show that the magnitude of NF-κB activation in 2D cultures is substantially higher than 3D cultures. However, unlike 2D cultures, cells in the 3D model remained responsive to TNF-α at higher concentrations. The more subdued and wider dynamic range of NF-κB responses in 3D culture system was associated with a different expression pattern for TNF receptor I in 3D versus 2D cultures collectively reflecting a more in vivo like TNF receptor I expression and NF-κB activation pattern in the 3D system. CONCLUSION: Our data suggest that lung fibroblasts are actively involved in the pathogenesis of lung inflammation by activation of NF-κB signaling pathway. The 3D culture detection system provides a sensitive and biologically relevant tool for investigating different pro-inflammatory events involving lung fibroblasts.

Immunocompetent 3D model of human upper airway for disease modeling and in vitro drug evaluation.

The development of more complex in vitro models for the assessment of novel drugs and chemicals is needed because of the limited biological relevance of animal models to humans as well as ethical considerations. Although some human-cell-based assays exist, they are usually 2D, consist of single cell type, and have limited cellular and functional representation of the native tissue. In this study, we have used biomimetic porous electrospun scaffolds to develop an immunocompetent 3D model of the human respiratory tract comprised of three key cell types present in upper airway epithelium. The three cell types, namely, epithelial cells (providing a physical barrier), fibroblasts (extracellular matrix production), and dendritic cells (immune sensing), were initially grown on individual scaffolds and then assembled into the 3D multicell tissue model. The epithelial layer was cultured at the air-liquid interface for up to four weeks, leading to formation of a functional barrier as evidenced by an increase in transepithelial electrical resistance (TEER) and tight junction formation. The response of epithelial cells to allergen exposure was monitored by quantifying changes in TEER readings and by assessment of cellular tight junctions using immunostaining. It was found that epithelial cells cocultured with fibroblasts formed a functional epithelial barrier at a quicker rate than single cultures of epithelial cells and that the recovery from allergen exposure was also more rapid. Also, our data show that dendritic cells within this model remain viable and responsive to external stimulation as evidenced by their migration within the 3D construct in response to allergen challenge. This model provides an easy to assemble and physiologically relevant 3D model of human airway epithelium that can be used for studies aiming at better understanding lung biology, the cross-talk between immune cells, and airborne allergens and pathogens as well as drug delivery.

Development and predictors of psychological adjustment during the course of community-based rehabilitation of traumatic brain injury: A preliminary study.

The aim of the study was to describe the development and predictors of psychological adjustment during community-based traumatic brain injury (TBI) rehabilitation. Forty-two adolescent and adult individuals with TBI (mean age 32 years, 88% male, median post-traumatic amnesia 11 days) participated in a single-group, longitudinal design study. The main measures used were the Reactions to Impairment and Disability Inventory, Adjustment subscale; Sydney Psychosocial Reintegration Scale; Hospital Anxiety and Depression Scale; and Self-awareness of Deficits Interview. At rehabilitation start, individuals differed significantly from each other with respect to their level of psychological adjustment. Individual trajectories of psychological adjustment were highly variable. However, for the sample as a whole, psychological adjustment did not change during the course of rehabilitation (multilevel regression models; p > .05). Good psychological adjustment was related to low levels of emotional distress and a small discrepancy between current and aspired functional status. Poor functional status had a more minor impact on psychological adjustment in individuals with poor self-awareness than in individuals with high levels of self-awareness. The results confirm parts of theoretical models of psychological adjustment to acquired brain injury, especially the importance of goal refinement. However, the results need to be treated cautiously, given the limited sample size.

About face: regional allied health professional early adaptation during the COVID-19 pandemic.

The coronavirus pandemic has led to significant change in allied health practice in Australia. Measures to slow virus spread have included replacing face-to-face services with telehealth services, and hands-on practice with socially distanced intervention. In the present mixed-methods, cross-sectional study, 51 allied health professionals across two public health services in regional Victoria, Australia, completed an online questionnaire with open and closed questions. The aim was to explore their experience in adapting to directed practice change during the first wave of the pandemic. The clinicians reported low levels of clinical satisfaction due to a perceived reduction in service quality and accessibility. Directed use of telehealth significantly contributed to dissatisfaction, with challenges including infrastructure, clinician and patient digital literacy and platform suitability for some patient groups and interventions. In contrast, peer support, timely and accurate communication, decision transparency, recognition and strong leadership from management supported adaptation, as did individuals' flexibility and learning. Our findings highlight the leadership qualities and support strategies conducive to workplace adaptation during a crisis period. They also support calls for further resource development to support skill translation for telehealth platform use and initiatives to increase digital literacy and infrastructure availability in regional Australia.

A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing.

Electrospinning is a common technique used to fabricate fibrous scaffolds for tissue engineering applications. There is now growing interest in assessing the ability of collector plate design to influence the patterning of the fibres during the electrospinning process. In this study, we investigate a novel method to generate hybrid electrospun scaffolds consisting of both random fibres and a defined three-dimensional (3D) micro-topography at the surface, using patterned resin formers produced by rapid prototyping (RP). Poly(D,L-lactide-co-glycolide) was electrospun onto the engineered RP surfaces and the ability of these formers to influence microfibre patterning in the resulting scaffolds visualized by scanning electron microscopy. Electrospun scaffolds with patterns mirroring the microstructures of the formers were successfully fabricated. The effect of the resulting fibre patterns and 3D geometries on mammalian cell adhesion and proliferation was investigated by seeding enhanced green fluorescent protein labelled 3T3 fibroblasts onto the scaffolds. Following 24 h and four days of culture, the seeded scaffolds were visually assessed by confocal macro- and microscopy. The patterning of the fibres guided initial cell adhesion to the scaffold with subsequent proliferation over the geometry resulting in the cells being held in a 3D micro-topography. Such patterning could be designed to replicate a specific in vivo structure; we use the dermal papillae as an exemplar here. In conclusion, a novel, versatile and scalable method to produce hybrid electrospun scaffolds has been developed. The 3D directional cues of the patterned fibres have been shown to influence cell behaviour and could be used to culture cells within a similar 3D micro-topography as experienced in vivo.

Self-reporting scaffolds for 3-dimensional cell culture.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biomimetic tissues on a chip for drug discovery.

Developing biologically relevant models of human tissues and organs is an important enabling step for disease modeling and drug discovery. Recent advances in tissue engineering, biomaterials and microfluidics have led to the development of microscale functional units of such models also referred to as 'organs on a chip'. In this review, we provide an overview of key enabling technologies and highlight the wealth of recent work regarding on-chip tissue models. In addition, we discuss the current challenges and future directions of organ-on-chip development.

The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent.

Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1-2, 1-3 and 1-6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.

Evaluation of a community-based model of rehabilitation following traumatic brain injury.

In recent years there has been a growing trend towards community-based post-acute rehabilitation for individuals with traumatic brain injury (TBI), as opposed to the traditional centre-based model, based on the premise that these individuals will learn more effectively in settings where they usually have to perform. In the present study, outcomes at two years post-injury in 77 individuals with TBI, treated within the community were compared on measures of activities of daily living (ADL), vocational status, and emotional adjustment with those of 77 TBI patients individually matched for gender, age, education, occupation, post-traumatic amnesia (PTA) duration, Glasgow Coma Scale (GCS) score and time in inpatient rehabilitation, who had attended the hospital for outpatient therapy. There were no significant differences between groups in terms of employment outcomes or independence in personal or domestic ADL. However those treated in the community were less likely to be independent in shopping and financial management and reported more changes in communication and social behaviour. Due to constraints of time and resources, these patients had received fewer one-on-one therapy sessions and thus treatment costs were somewhat lower. Attendant care costs were also lower in the community treatment group. Strengths and weaknesses of community-based post-acute rehabilitation are discussed.