Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles.

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.

Patient journeys: the process of clinical redesign.

*Clinical process redesign is a successful improvement method that has been used to increase access to health services in 60 public hospitals across New South Wales, and at Flinders Medical Centre (FMC) in South Australia. *The method focuses on the patient journey as the primary improvement locus, and uses process mapping to identify the value-adding steps in that journey; it involves redesign teams identifying and eliminating non-value-adding steps to improve flow and reduce delays in access to emergency and elective care. *The method engages clinicians, managers, patients and carers, and delivers real gains in health care delivery. *This article outlines the clinical process redesign programs being used by NSW Health and at FMC.

Clinical process redesign for unplanned arrivals in hospitals.

*Emergency department performance had been deteriorating in NSW Health facilities and at Flinders Medical Centre before a fundamentally new approach involving a redesign method, additional bed capacity and more rigorous hospital performance management was applied. *Redesign was undertaken in over 60 hospitals in New South Wales. *Numerous disconnections and misalignments in the process of care delivery have been uncovered during the diagnostic phase of this redesign. *Solutions addressed the entire patient journey through the hospital, to produce smoother patient flow along the continuum of care. *To achieve a sustained improvement in performance, numerous solutions must be simultaneously implemented in each hospital. *With this multipronged approach, a turnaround in NSW emergency access performance has been achieved in the face of rising demand for services; the improvement has continued over 3 years. *This article reports on our findings from system-wide redesign for unplanned hospital attendances.

Applying clinical process redesign methods to planned arrivals in New South Wales hospitals.

*Competing demands of planned and unplanned arrivals present major challenges for hospitals. *Applying clinical process redesign methods to the planned patient journey allows management to recognise the blocks and inefficiencies in the journey and facilitates the development of solutions for improvement. *Redesign of the planned patient journey in New South Wales has promoted the expansion of the extended day-only model of care, reformed the waiting times policy, standardised patient preadmission assessment and preparation, and targeted operating theatre use. *Improved performance management at Area Health Service and local facility levels has accompanied the redesign of planned arrival processes. *The results in redesign of surgery undertaken by the Area Health Services in 96 NSW hospitals have been impressive, with results within 2 years of commencing the clinical services redesign program showing: a 97% reduction in the numbers of patients in Category 1 (admission desirable within 30 days) whose surgery was overdue, from 5308 in January 2005 to 135 in June 2007; and a 99% reduction in the number of patients who have waited > 365 days for surgery, from 10 551 in January 2005 to 84 in June 2007. *Improved surgical service efficiency, safety and quality justify the continuation of the redesign program.

Implementing and sustaining transformational change in health care: lessons learnt about clinical process redesign.

*Clinical process redesign has enabled significant improvements in the delivery of health care services in emergency departments and elective surgery programs in New South Wales and at Flinders Medical Centre in South Australia, with tangible benefits for patients and staff. *The principles used in clinical process redesign are not new; they have been applied in other industries with significant gains for many years, but have only recently been introduced into health care systems. *Through experience with clinical process redesign, we have learnt much about the factors critical to the success of implementing and sustaining this process in the health care setting. *The key elements for success are leadership by senior executives, clinical leadership, team-based problem solving, a focus on the patient journey, access to data, ambitious targets, strong performance management, and a process for maintaining improvement.

Health services under siege: the case for clinical process redesign.

*Public health services are struggling to cope with rising demand. *Strain on health services manifests as longer waiting lists for surgery, queuing in the emergency departments, increased rates of adverse events, and delays in discharge, particularly for older patients. *Traditional responses are not resolving these problems. *Analysis shows that the day-to-day system processes underlying clinical care are poorly designed and do not produce a well planned, well coordinated patient journey. *Numerous disconnections along the continuum of care have a cumulative effect in obstructing patient flow and causing frustration for patients and staff. *Rigorous clinical process redesign methods can significantly improve performance, even in the face of rising demand.

Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.