Imaging actin organisation and dynamics in 3D.

The actin cytoskeleton plays a critical role in cell architecture and the control of fundamental processes including cell division, migration and survival. The dynamics and organisation of F-actin have been widely studied in a breadth of cell types on classical two-dimensional (2D) surfaces. Recent advances in optical microscopy have enabled interrogation of these cytoskeletal networks in cells within three-dimensional (3D) scaffolds, tissues and in vivo. Emerging studies indicate that the dimensionality experienced by cells has a profound impact on the structure and function of the cytoskeleton, with cells in 3D environments exhibiting cytoskeletal arrangements that differ to cells in 2D environments. However, the addition of a third (and fourth, with time) dimension leads to challenges in sample preparation, imaging and analysis, necessitating additional considerations to achieve the required signal-to-noise ratio and spatial and temporal resolution. Here, we summarise the current tools for imaging actin in a 3D context and highlight examples of the importance of this in understanding cytoskeletal biology and the challenges and opportunities in this domain.

A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

Occurrence of adverse events and change in disease activity after initiation of etanercept in paediatric patients with juvenile psoriatic arthritis in the CARRA Registry.

OBJECTIVE: Etanercept is commonly used to treat juvenile idiopathic arthritis, including juvenile psoriatic arthritis (JPsA); however, information on etanercept's safety and effectiveness in clinical practice is limited. We used data from the Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry to evaluate etanercept's safety and effectiveness in JPsA in clinical practice. METHODS: We analysed safety and effectiveness data for paediatric patients enrolled in the CARRA Registry who had a JPsA diagnosis and had used etanercept. Safety was assessed by calculating rates of prespecified adverse events of special interest (AESIs) and serious adverse events (SAEs). Effectiveness was assessed by a variety of disease activity measures. RESULTS: Overall, 226 patients had JPsA and received etanercept; 191 met criteria for safety analysis and 43 met criteria for effectiveness analysis. AESI and SAE incidence rates were low. There were five events: three uveitis, one new-onset neuropathy and one malignancy. Incidence rates were 0.55 (95% CI: 0.18, 1.69), 0.18 (95% CI: 0.03, 1.29) and 0.13 (95% CI: 0.02, 0.09) per 100 patient-years for uveitis, neuropathy and malignancy, respectively. Etanercept showed effectiveness for JPsA treatment; 7 of 15 (46.7%) had an American College of Rheumatology-Pediatric Response 90, 9 of 25 (36.0%) had a clinical Juvenile Arthritis Disease Activity Score 10-joint ≤1.1 and 14 of 27 (51.9%) had clinically inactive disease at the 6-month follow-up. CONCLUSION: Data in the CARRA Registry showed that etanercept treatment was safe in treating children with JPsA, with low AESIs and SAEs. Etanercept was also effective, even when assessed in a small sample size.

Disease Recapture Rates After Medication Discontinuation and Flare in Juvenile Idiopathic Arthritis: An Observational Study Within the Childhood Arthritis and Rheumatology Research Alliance Registry.

OBJECTIVE: Children with well-controlled juvenile idiopathic arthritis (JIA) frequently experience flares after medication discontinuation, but the outcomes of these flares have not been well described. The objective of this study was to characterize the rates and predictors of disease recapture among children with JIA who restarted medication to treat disease flare. METHODS: Children with JIA who discontinued conventional synthetic or biologic disease-modifying antirheumatic drugs for well-controlled disease but subsequently experienced a flare and restarted medication were identified from the Childhood Arthritis and Rheumatology Research Alliance (CARRA) registry. The primary outcome was inactive disease (ID) (physician global assessment <1 and active joint count = 0) 6 months after flare. RESULTS: A total of 333 patients had complete data for ID at 6 months after flare. The recapture rate for the cohort was 55%, ranging from 47% (persistent oligoarthritis) to 69% (systemic arthritis) (P = 0.4). Approximately 67% of children achieved ID by 12 months. In the multivariable model, history and reinitiation of biologic drugs were associated with increased odds of successful recapture (odds ratio [OR] 4.79 [95% confidence interval (95% CI) 1.22-18.78] and OR 2.74 [95% CI 1.62-4.63], respectively). Number of joints with limited range of motion was associated with decreased odds (OR 0.83 per 1 joint increase [95% CI 0.72-0.95]). CONCLUSION: Approximately half of JIA flares post-discontinuation were recaptured within 6 months, but rates of recapture varied across JIA categories. These findings inform shared decision-making for patients, families, and clinicians regarding the risks and benefits of medication discontinuation. Better understanding of biologic predictors of successful recapture in JIA are needed.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.

Evaluating Foundational Data Quality in the National Patient-Centered Clinical Research Network (PCORnet®).

INTRODUCTION: Distributed research networks (DRNs) are critical components of the strategic roadmaps for the National Institutes of Health and the Food and Drug Administration as they work to move toward large-scale systems of evidence generation. The National Patient-Centered Clinical Research Network (PCORnet®) is one of the first DRNs to incorporate electronic health record data from multiple domains on a national scale. Before conducting analyses in a DRN, it is important to assess the quality and characteristics of the data. METHODS: PCORnet's Coordinating Center is responsible for evaluating foundational data quality, or assessing fitness-for-use across a broad research portfolio, through a process called data curation. Data curation involves a set of analytic and querying activities to assess data quality coupled with maintenance of detailed documentation and ongoing communication with network partners. The first cycle of PCORnet data curation focused on six domains in the PCORnet common data model: demographics, diagnoses, encounters, enrollment, procedures, and vitals. RESULTS: The data curation process led to improvements in foundational data quality. Notable improvements included the elimination of data model conformance errors; a decrease in implausible height, weight, and blood pressure values; an increase in the volume of diagnoses and procedures; and more complete data for key analytic variables. Based on the findings of the first cycle, we made modifications to the curation process to increase efficiencies and further reduce variation among data partners. DISCUSSION: The iterative nature of the data curation process allows PCORnet to gradually increase the foundational level of data quality and reduce variability across the network. These activities help increase the transparency and reproducibility of analyses within PCORnet and can serve as a model for other DRNs.

Pilot study comparing the childhood arthritis and rheumatology research alliance consensus treatment plans for induction therapy of juvenile proliferative lupus nephritis.

BACKGROUND: To reduce treatment variability and facilitate comparative effectiveness studies, the Childhood Arthritis and Rheumatology Research Alliance (CARRA) published consensus treatment plans (CTPs) including one for juvenile proliferative lupus nephritis (LN). Induction immunosuppression CTPs outline treatment with either monthly intravenous (IV) cyclophosphamide (CYC) or mycophenolate mofetil (MMF) in conjunction with one of three corticosteroid (steroid) CTPs: primarily oral, primarily IV or mixed oral/IV. The acceptability and in-practice use of these CTPs are unknown. Therefore, the primary aims of the pilot study were to demonstrate feasibility of adhering to the LN CTPs and delineate barriers to implementation in clinical care in the US. Further, we aimed to explore the safety and effectiveness of the treatments for induction therapy. METHODS: Forty-one patients were enrolled from 10 CARRA sites. Patients had new-onset biopsy proven ISN/RPS class III or IV proliferative LN, were starting induction therapy with MMF or IV CYC and high-dose steroids and were followed for up to 24 months. Routine clinical data were collected at each visit. Provider reasons for CTP selection were assessed at baseline. Adherence to the CTPs was evaluated by provider survey and medication logs. Complete and partial renal responses were reported at 6 months. RESULTS: The majority of patients were female (83%) with a mean age of 14.7 years, SD 2.8. CYC was used more commonly than MMF for patients with ISN/RPS class IV LN (vs. class III), those who had hematuria, and those with adherence concerns. Overall adherence to the immunosuppression induction CTPs was acceptable with a majority of patients receiving the target MMF (86%) or CYC (63%) dose. However, adherence to the steroid CTPs was poor (37%) with large variability in dosing. Renal response endpoints were exploratory and did not show a significant difference between CYC and MMF. CONCLUSIONS: Overall, the immunosuppression CTPs were followed as intended in the majority of patients however, adherence to the steroid CTPs was poor indicating revision is necessary. In addition, our pilot study revealed several sources of treatment selection bias that will need to be addressed in for future comparative effectiveness research.