Differential proteolytic activation of the Bacillus thuringiensis Cry41Aa parasporin modulates its anticancer effect.

Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells) cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made in the N-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore, we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60, respectively) which we hypothesize occlude a binding motif.

The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts.

Understanding how certain protein toxins from the normally insecticidal bacterium Bacillus thuringiensis (Bt) target human cell lines has implications for both the risk assessment of products containing these toxins and potentially for cancer therapy. This understanding requires knowledge of whether the human cell active toxins work by the same mechanism as their insecticidal counterparts or by alternative ones. The Bt Cry41Aa (also known as Parasporin3) toxin is structurally related to the toxins synthesised by commercially produced transgenic insect-resistant plants, with the notable exception of an additional C-terminal ß-trefoil ricin domain. To better understand its mechanism of action, we developed an efficient expression system for the toxin and created mutations in regions potentially involved in the toxic mechanism. Deletion of the ricin domain did not significantly affect the activity of the toxin against the human HepG2 cell line, suggesting that this region was not responsible for the mammalian specificity of Cry41Aa. Various biochemical assays suggested that unlike some other human cell active toxins from Bt Cry41Aa did not induce apoptosis, but that its mechanism of action was consistent with that of a pore-forming toxin. The toxin induced a rapid and significant decrease in metabolic activity. Adenosine triphosphate depletion, cell swelling and membrane damage were also observed. An exposed loop region believed to be involved in receptor binding of insecticidal Cry toxins was shown to be important for the activity of Cry41Aa against HepG2 cells.

Usability of the zilucoplan prefilled syringe for the treatment of generalized myasthenia gravis: insights from a human factors validation study.

BACKGROUND: Generalized myasthenia gravis (gMG) is a chronic, autoimmune neuromuscular disease, often accompanied by high treatment burden. The objective of this usability study was to validate that the zilucoplan prefilled syringe (PFS) can be used safely and effectively. METHODS: The study recruited 75 participants representing five distinct user groups (injection-naïve gMG patients, injection-experienced gMG patients, injection-naïve caregivers, injection-experienced caregivers, and healthcare professionals), who each simulated an injection without being directed to use the instructions for use (IFU). Participants were then asked to repeat the process as directed by the IFU. Participants were assessed and use errors identified using performance-based criteria. RESULTS: 73/75 (97.3%) participants were able to safely administer the dose of medication during the first simulated use, with or without the IFU. When utilizing the IFU, all 75 (100.0%) participants were able to administer the medication. The rate of use errors was low, with pass rates for observation-based tasks ranging from 85.3% to 100.0%, and in general, after participants were instructed to refer to the IFU, the rate of use errors was reduced. CONCLUSIONS: This human factors study demonstrated that the zilucoplan PFS is safe and effective for intended users, including patients with gMG and their caregivers.

Probing the Mechanism of Action of Cry41Aa on HepG2 through the Establishment of a Resistant Subline.

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium Bacillus thuringiensis that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism.

A Companion App to Support Rheumatology Patients Treated with Certolizumab Pegol: Results From a Usability Study.

BACKGROUND: Certolizumab pegol (CZP) is an anti-tumor necrosis factor drug approved for the treatment of multiple moderate to severe chronic inflammatory diseases. In the European Union, CZP is approved for administration by subcutaneous self-injection using a prefilled syringe, prefilled pen, or reusable electromechanical auto-injector (electronic device). CimplyMe is a companion app for use alongside CZP self-injection devices, designed to support CZP-treated patients self-managing their treatment and disease. OBJECTIVE: This study aimed to validate the usability of the companion app by demonstrating that tasks required for use can be performed successfully by intended end users. METHODS: We recruited 15 patients with moderate to severe rheumatoid arthritis, currently prescribed biologic treatment, and using apps on a smart phone. Patients were assessed on their ability to use the companion app in a setting designed to simulate a location where patients regularly administer biologic treatment. To assess the usability of the key features of the app, 8 critical and 3 noncritical tasks were designed. Patients' success on each task was recorded through observations or knowledge-based questions. Successes with difficulty and use errors were also recorded. If a patient made a use error at the first attempt, a second attempt was allowed. Second-attempt use errors were recorded as a task failure. RESULTS: A total of 207 first attempts at the 14 components of the 8 critical tasks were evaluated (3 patients failed to complete one component); 178 (86.0%) critical tasks were successfully completed at the first attempt. The remaining first attempts comprised 16 (7.7%) successes with difficulty and 13 (6.3%) use errors, which had to be repeated. One critical task was not re-attempted by one patient due to time constraints; however, there were no use errors in the 12 completed second attempts. A total of 107 first attempts at the 3 noncritical tasks were made, all of which (107/107, 100.0%) were completed without use errors. CONCLUSIONS: In simulated testing, patients were able to successfully use the companion app without formal training. This study suggests the companion app is easy to use and could help patients prescribed CZP better manage their treatment and disease.

The role of membrane-bound metal ions in toxicity of a human cancer cell-active pore-forming toxin Cry41Aa from Bacillus thuringiensis.

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca2+, Mn2+ and Zn2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action.

A portfolio of biologic self-injection devices in rheumatology: how patient involvement in device design can improve treatment experience.

Biologic drugs (e.g. anti-tumor necrosis factors) are effective treatments for multiple chronic inflammatory diseases including rheumatoid arthritis, axial spondyloarthritis, and psoriatic arthritis. Administration of biologic drugs is usually via subcutaneous self-injection, which provides many patient benefits compared to infusions including increased flexibility, reduced costs, and reduced caregiver burden. However, it is also associated with challenges such as needle phobia, patient treatment misconceptions and incorrect drug administration, and can be impacted by dexterity problems. Evidence suggests these problems, along with other drug administration challenges (e.g. patient forgetfulness, busy lifestyles, and polypharmacy), can reduce patient adherence to treatment. To combat these challenges, patient feedback has been used to develop a range of self-injection devices, including pre-filled syringes, pre-filled pens, and electronic injection devices. Providing different devices for drug administration gives patients the opportunity to choose a device that addresses the challenges they face as an individual. Research suggests involving patients in medical device development, providing patients with a choice of devices and enrolling individuals in patient support programs can empower patients to take control of their treatment journey. By providing a portfolio of self-injection devices, designed based on patient needs, patient experience will improve, potentially improving adherence and hence, long-term treatment outcomes.

Using Patient Feedback to Optimize the Design of a Certolizumab Pegol Electromechanical Self-Injection Device: Insights from Human Factors Studies.

INTRODUCTION: We incorporated patient feedback from human factors studies (HFS) in the patient-centric design and validation of ava®, an electromechanical device (e-Device) for self-injecting the anti-tumor necrosis factor certolizumab pegol (CZP). METHODS: Healthcare professionals, caregivers, healthy volunteers, and patients with rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or Crohn's disease participated in 11 formative HFS to optimize the e-Device design through intended user feedback; nine studies involved simulated injections. Formative participant questionnaire feedback was collected following e-Device prototype handling. Validation HFS (one EU study and one US study) assessed the safe and effective setup and use of the e-Device using 22 predefined critical tasks. Task outcomes were categorized as "failures" if participants did not succeed within three attempts. RESULTS: Two hundred eighty-three participants entered formative (163) and validation (120) HFS; 260 participants performed one or more simulated e-Device self-injections. Design changes following formative HFS included alterations to buttons and the graphical user interface screen. All validation HFS participants completed critical tasks necessary for CZP dose delivery, with minimal critical task failures (12 of 572 critical tasks, 2.1%, in the EU study, and 2 of 5310 critical tasks, less than 0.1%, in the US study). CONCLUSION: CZP e-Device development was guided by intended user feedback through HFS, ensuring the final design addressed patients' needs. In both validation studies, participants successfully performed all critical tasks, demonstrating safe and effective e-Device self-injections. FUNDING: UCB Pharma. Plain language summary available on the journal website.

Nurse- and peer-led self-management programme for patients with an implantable cardioverter defibrillator; a feasibility study.

BACKGROUND: The prevalence of cardiovascular disease is increasing. Improved treatment options increase survival after an acute myocardial infarction or sudden cardiac arrest, although patients often have difficulty adjusting and regaining control in daily life. In particular, patients who received an implantable cardioverter defibrillator (ICD) experience physical and psychological problems. Interventions to enhance perceived control and acceptance of the device are therefore necessary. This paper describes a small-scale study to explore the feasibility and the possible benefits of a structured nurse- and peer-led self-management programme ('Chronic Disease Self-Management Program' - CDSMP) among ICD patients. METHODS: Ten male ICD patients (mean age = 65.5 years) participated in a group programme, consisting of six sessions, led by a team consisting of a nurse specialist and a patient with cardiovascular disease. Programme feasibility was evaluated among patients and leaders by measuring performance of the intervention according to protocol, attendance and adherence of the participating ICD patients, and patients' and leaders' opinions about the programme. In addition, before and directly after attending the intervention, programme benefits (e.g. perceived control, symptoms of anxiety and depression, and quality of life) were assessed. RESULTS: The programme was conducted largely according to protocol. Eight patients attended at least four sessions, and adherence ranged from good to very good. On average, the patients reported to have benefited very much from the programme, which they gave an overall report mark of 8.4. The leaders considered the programme feasible as well. Furthermore, improvements were identified for general self-efficacy expectancies, symptoms of anxiety, physical functioning, social functioning, role limitations due to physical problems, and pain. CONCLUSION: This study suggests that a self-management programme led by a team consisting of a nurse specialist and a patient with cardiovascular disease seems feasible according to both patients and leaders. The programme may improve general self-efficacy expectancies, symptoms of anxiety, and quality of life (physical functioning, social functioning, role limitations due to physical problems, and pain) as well. Further investigation of the programme's effectiveness among a larger sample of ICD patients or other patient groups with cardiovascular disease, is recommended.

Comparative usability study for a certolizumab pegol autoinjection device in patients with rheumatoid arthritis.

OBJECTIVES: To compare the usability of a new certolizumab pegol (CZP) autoinjector with the adalimumab, etanercept, and golimumab devices in patients with rheumatoid arthritis. METHODS: Two identical studies were performed in 2013 and 2016; patients performed a simulated self-injection with the CZP autoinjector and the most up-to-date device versions at the time in a randomized, consecutive sequence. The primary end point was the ranking of the four autoinjectors in order of preference. Device usability and intuitiveness were assessed across a range of secondary and exploratory end points. RESULTS: The 2013 and 2016 study populations included 76 patients each; a significant majority (2013: 67%; 2016: 59%) ranked the CZP autoinjector as their most preferred device (p < 0.001). Most patients agreed that the CZP autoinjector was easier to use, start, and manipulate, and were more willing to use it than the comparator devices (p < 0.001 for all pairwise comparisons with CZP). Likert score differences also favored the CZP autoinjector regarding how easy it was to determine injection completion. The CZP autoinjector was associated with a low rate of use error. CONCLUSIONS: In both studies, the CZP autoinjector was the preferred choice compared to the alternative devices and was associated with a high level of patient satisfaction.

The effect of a new syringe design on the ability of rheumatoid arthritis patients to inject a biological medication.

Self-administration of new biological medications can be difficult for Rheumatoid Arthritis patients with functional impairment and hand and dexterity limitation. Twenty-three Rheumatoid Arthritis (RA) patients participated in this study to compare preferences and injection forces using a conventional syringe and a new ergonomically designed syringe. Injection force measurements were collected in two ways: a) isometric forces, with the syringes' plungers in fixed positions (depressed halfway and fully depressed), and b) forces exerted during injection of the medication. Subjects' grip and pinch strengths were measured. A perception questionnaire gauged subjects' impressions and preferences. Subjects were capable of exerting significantly higher isometric forces using the new syringe with the plunger fixed both halfway and fully depressed. During injection of the medication, peak and mean injection forces were significantly higher, and duration was shorter, when using the new syringe. Subjects rated the new syringe higher on all twenty attributes on preference and performance. Therefore, it is expected that the new syringe will benefit self-administration of medication injection for RA patients.