Priorities for research in multiple conditions in later life (multi-morbidity): findings from a James Lind Alliance Priority Setting Partnership.

INTRODUCTION: multiple conditions in later life (multi-morbidity) is a major challenge for health and care systems worldwide, is of particular relevance for older people, but has not (until recently) received high priority as a topic for research. We have identified the top 10 research priorities from the perspective of older people, their carers, and health and social care professionals using the methods of a James Lind Alliance Priority Setting Partnership. METHODS: in total, 354 participants (162 older people and carers, 192 health professionals) completed a survey and 15 older people and carers were interviewed to produce 96 'unanswered questions'. These were further refined by survey and interviews to a shortlist of 21 topics, and a mix of people aged 80+ living with three or more conditions, carers and health and social care providers to prioritised the top 10. RESULTS: the key priorities were about the prevention of social isolation, the promotion of independence and physical and emotional well-being. In addition to these broad topics, the process also identified detailed priorities including the role of exercise therapy, the importance of falls (particularly fear of falling), the recognition and management of frailty and Comprehensive Geriatric Assessment. CONCLUSION: these topics provide a unique perspective on research priorities on multiple conditions in later life and complement existing UK and International recommendations about the optimisation of health and social care systems to deliver essential holistic models of care and the prevention and treatment of multiple co-existing conditions.

Fatigue severity remains stable over time and independently associated with orthostatic symptoms in chronic fatigue syndrome: a longitudinal study.

OBJECTIVES: to examine fatigue variability over time in chronic fatigue syndrome (CFS) and the effect of other symptoms on its predictability. DESIGN: longitudinal cohort study of patients with CFS (Fukuda criteria). SETTING: specialist CFS clinical service. SUBJECTS: phase 1: 100 patients who participated in a study of CFS symptoms in 2005 were revisited in 2009. Phase 2: 25 patients completed fatigue diaries to address intra- and inter-day variability in perceived fatigue. MAIN OUTCOME MEASURES: phase 1: subjects completed fatigue impact scale (FIS), Epworth sleepiness scale (ESS), orthostatic grading scale (OGS) and hospital anxiety and depression scale (HADS). Changes in variables represented the differences between 2005 and 2009. Phase 2: subjects rated fatigue on a scale of 0 (no fatigue) to 10 (severe fatigue) four times a day for 5 weeks. RESULTS: symptom assessment tools were available in both 2005 and 2009 for 74% of patients. FIS and HADS depression (HAD-D) and anxiety (HAD-A) scores significantly improved during follow-up whereas ESS and OGS remained stable. FIS improved in 29/74 (39%) subjects, and by ≥ 10 points in 19 (26%). FIS worsened by ≥ 10 points in 33/74 (45%) subjects. On multivariate analysis, independent predictors of current fatigue (FIS in 2009) were FIS in 2005, HAD-D in 2009, OGS in 2009 and change in HAD-A. Reported fatigue was stable from week to week and from day to day. Patients reported higher fatigue in the morning (mean ± SD; 6.4 ± 2), becoming significantly lower at lunchtime (6.2 ± 2; P < 0.05) and increasing again to 7 ± 2 at bedtime. CONCLUSIONS: current fatigue is independently associated with current autonomic symptom burden, current depression and change in anxiety during follow-up. These findings have implications for targeted symptom management in CFS.

The complexities of defining atopy in severe childhood asthma.

BACKGROUND: Defining atopy in children with severe, therapy-resistant asthma is complex. There is currently no gold standard test; both skin prick testing (SPT) and allergen-specific IgE (sIgE) are used. Furthermore, atopy is increasingly considered to be a spectrum, not an all-or-none phenomenon. HYPOTHESIS: SPTs and sIgE cannot be used interchangeably, and if both tests are not performed, opportunities for intervention will be missed. Furthermore, the severity of atopy will be defined differently by the two tests. METHODS: Cross-sectional study of 47 children with severe, therapy-resistant asthma, mean age 11.8 years, range 5.3-16.6 years, who underwent SPT, and measurement of total and sIgE as part of their clinical work-up. RESULTS: Overall, 42/47 (89%) were atopic (defined as either one positive SPT or sIgE). There was 98% concordance between the two tests in classifying atopy. When each allergen was considered individually, in 40/200 (20%), the SPT and sIgE results were discordant, most commonly in 25/200 (12.5%), the SPT was negative and the sIgE was positive. House dust mite and cat sensitization were more likely detected by sIgE, but dog sensitization by SPT. When atopy was quantified, the sum of sIgEs compared with the sum of SPT weal diameter showed a moderate correlation (r(2) =0.44, P<0.001). Total IgE increased with an increasing number of positive sIgEs (P=0.028), but not significantly with increasing numbers of positive SPTs. CONCLUSION AND CLINICAL RELEVANCE: SPT and sIgE identify group prevalence of atopy equally well; however, for individual allergens, concordance is poor, and when used to quantify atopy, SPTs and sIgE were only moderately correlated. In a clinical setting, if allergen avoidance is contemplated in children with severe, therapy-resistant asthma, both tests should be performed in order to detect sensitization.

Printability and bio-functionality of a shear thinning methacrylated xanthan-gelatin composite bioink.

3D bioprinting is a recent technique that can create complex cell seeded scaffolds and therefore holds great promise to revolutionize the biomedical sector by combining materials and structures that more closely mimic the 3D cell environment in tissues. The most commonly used biomaterials for printing are hydrogels, however, many of the hydrogels used still present issues of printability, stability, or poor cell-material interactions. We propose that bioinks with intrinsic self-assembling and shear thinning properties, such as xanthan gum, can be methacrylated (XGMA) and combined with a bio-functional material such as gelatin methacryloyl (GelMa) to create a stable, cell-interactive bioink with improved properties for 3D bioprinting. These biomaterials have reduced viscosity under high shear and recover their viscosity rapidly after the shear is removed, retaining their shape, which translates to easier extrusion whilst maintaining accurate fidelity after printing. This was confirmed in printing studies, with measured normalized strand widths of 1.2 obtained for high gel concentrations (5+5 % XGMA-GelMA). Furthermore, the introduction of a secondary photo-cross-linking method allowed tuning of the mechanical properties of the hydrogel with stiffness between 15 and 30 kPa, as well as improving the stability of the hydrogel with retention of 75 % of its mass after 90 d. The hydrogel was shown to be biocompatible and bio-active with 97 % cell viability, and cell spreading after 7 d of culture for low gel concentrations (3+3 % XGMA-GelMA). Shear stresses were relatively low while printing (1 kPa) as a result of the shear thinning property of the material, which supported cell viability during extrusion. Finally, printed hydrogels retained high cell viability for lower gel concentrations, and showed improved cell viability for more concentrated hydrogels when compared to cells cultured in bulk hydrogels, presumably due to improved nutrient/oxygen diffusion and cell migration. In conclusion, stability and formulation of a XGMA-GelMA shear thinning composite hydrogel has been optimized to create a bio-functional bioink, with improved printability, andin vitroculture stabilityviasecondary photo-induced cross-linking, making this composite a promising bioink for 3D bioprinting.

In situ miRNA delivery from a hydrogel promotes osteogenesis of encapsulated mesenchymal stromal cells.

Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100-5p/miR-143-3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering.

Polyethylene glycol-gelatin hydrogels with tuneable stiffness prepared by horseradish peroxidase-activated tetrazine-norbornene ligation.

Tetrazine-norbornene ligation has previously been applied in bioorthognal polymer crosslinking to form hydrogels suitable for 3D cell culture. However, the tetrazine group is prone to reduction by the free thiol in a biological environment, reducing the crosslinking efficiency and shortening the storage of tetrazine containing linkers. Here, we introduce a method to form a tetrazine group in situ by catalytic oxidation of the dihydrogen tetrazine using horse radish peroxidase (HRP). Enzymatic oxidation is highly efficient at a low HRP concentration and does not require hydrogen peroxide, allowing for rapid gelation when HRP was added to an aqueous solution of 4-arm PEG dihydrogentetrazine and gelatin norbornene. The storage modulus of the resultant gels can be varied by changing the concentration of the crosslinker, which is in the range of 1.2-3.8 kPa. Human mesenchymal stem cells encapsulated within these gels, with varying stiffness, display varied interactions and morphologies and can be maintained with prolonged culture periods of at least 32 days of 3D culture. The enzymatic activation of tetrazine-norbornene is therefore an attractive addition to the tetrazine ligation that is highly suitable for cell related studies in tissue engineering.

The duration of the orthostatic blood pressure drop is predictive of death.

BACKGROUND: Orthostatic hypotension (OH) affects 6% of community-dwelling older people. This increases to 60% when non-invasive, continuous blood pressure (BP) monitoring is used, due to identification of transient drops in BP which recover rapidly. AIM: To determine the clinical relevance of these transient orthostatic BP drops. DESIGN: Five-year clinical observational study. METHODS: One hundred three consecutive new patients attending a Falls and Syncope Clinic in the UK from 1 February 2009 underwent continuous BP monitoring during an active stand. BP profiles were analysed to quantify all reductions in BP, measuring the duration of any drop below diagnostic criteria. Five-year follow-up data were extracted from hospital clinical records to assess clinical outcomes. RESULTS: Systolic BP (sBP) dropped ≥20 mmHg in 76 (74%) individuals, with 65 (63%) having ≥10 mmHg drop in diastolic BP. However, only 22 (21%) cases were diagnosed clinically with OH. A sustained reduction in BP (≥30 s) had a sensitivity of 0.91 and specificity of 0.88 for a clinical diagnosis of OH, being more accurate than absolute BP reduction alone. A sustained reduction in sBP was associated with greater use of vasopressors (36%,P0.001) and an independent, significantly greater risk of death (45% at 5 years,P0.009). CONCLUSION: An orthostatic reduction in sBP lasting ≥30 s improves accuracy of diagnosis. Moreover, given the significant adverse outcomes with a sustained reduction, clinicians should consider this when diagnosing and treating patients, as transient OH does not appear to be clinically significant.

A new method to prevent degradation of lithium-oxygen batteries: reduction of superoxide by viologen.

Lithium-oxygen battery development is hampered by degradation reactions initiated by superoxide, which is formed in the pathway of oxygen reduction to peroxide. This work demonstrates that the superoxide lifetime is drastically decreased upon addition of ethyl viologen, which catalyses the reduction of superoxide to peroxide.

Effects of electric fields on human mesenchymal stem cell behaviour and morphology using a novel multichannel device.

The intrinsic piezoelectric nature of collagenous-rich tissues, such as bone and cartilage, can result in the production of small, endogenous electric fields (EFs) during applied mechanical stresses. In vivo, these EFs may influence cell migration, a vital component of wound healing. As a result, the application of small external EFs to bone fractures and cutaneous wounds is actively practiced clinically. Due to the significant regenerative potential of stem cells in bone and cartilage healing, and their potential role in the observed improved healing in vivo post applied EFs, using a novel medium throughput device, we investigated the impacts of physiological and aphysiological EFs on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for up to 15 hours. The applied EFs had significant impacts on hBM-MSC morphology and migration; cells displayed varying degrees of conversion to a highly elongated phenotype dependent on the EF strength, consistent perpendicular alignment to the EF vector, and definitive cathodal migration in response to EF strengths ≥0.5 V cm(-1), with the fastest migration speeds observed at between 1.7 and 3 V cm(-1). We observed variability in hBM-MSC donor-to-donor responses and overall tolerances to applied EFs. This study thus confirms hBM-MSCs are responsive to applied EFs, and their rate of migration towards the cathode is controllable depending on the EF strength, providing new insight into the physiology of hBM-MSCs and possibly a significant opportunity for the utilisation of EFs in directed scaffold colonisation in vitro for tissue engineering applications or in vivo post implantation.

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro.

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(beta, gamma-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.

The pharmacology of bisphosphonates and new insights into their mechanisms of action.

Bisphosphonates are chemically stable analogs of inorganic pyrophosphate, which are resistant to breakdown by enzymatic hydrolysis. The biological effects of bisphosphonates on calcium metabolism were originally ascribed to their physico-chemical effects on hydroxyapatite crystals. Although such effects may contribute to their overall action, their effects on cells are probably of greater importance, particularly for the more potent compounds. Remarkable progress has been made in increasing the potency of bisphosphonates as inhibitors of bone resorption, and the most potent compounds in current use are characterized by the presence of a nitrogen atom at critical positions in the side chain which, together with the bisphosphonate moiety itself, seems to be essential for maximal activity. As a class the bisphosphonates offer a very effective means of treating Paget's disease.

Contrasting effects of alendronate and clodronate on RAW 264 macrophages: the role of a bisphosphonate metabolite.

Clodronate (dichloromethylidene-bisphosphonate), a halogen-containing bisphosphonate, can inhibit the release of cytokines from RAW 264 macrophages and has anti-inflammatory properties in rheumatoid arthritis, whilst amino-containing bisphosphonates such as alendronate (4-amino-1-hydroxybutylidene-bisphosphonate), have pro-inflammatory properties and can cause an acute phase response. The basis for these pharmacological properties is unclear. Recently, it was demonstrated that clodronate is metabolised by certain cell lines in vitro to an analogue of ATP, whereas amino-bisphosphonates are not. We therefore investigated whether clodronate can also be metabolised by RAW 264 macrophages and whether intracellular accumulation of the metabolite (AppCCl2p) could account for the anti-inflammatory properties of clodronate. The effect of alendronate and AppCCl2p on the release of cytokines (IL-1beta, IL-6, and TNFalpha) from RAW 264 cells was compared, and the effect of the bisphosphonates and AppCCl2p on the DNA binding activities of transcription factors, NF-kappaB and AP-1, was investigated. Pretreatment of RAW 264 macrophages with alendronate augmented the LPS-stimulated release of IL-1beta and increased the binding of NF-kappaB to DNA in an electrophoretic mobility shift assay. Without LPS-induction, alendronate did not affect cytokine release or NF-kappaB binding. Clodronate was metabolised by RAW 264 cells to AppCCl2p. Like clodronate, AppCCl2p inhibited the LPS-induced release of cytokines and NO from RAW 264 macrophages. Both clodronate and its metabolite also inhibited the LPS-stimulated binding of NF-kappaB to DNA. In conclusion, these results suggest that the metabolite of clodronate may be responsible for the anti-inflammatory properties of clodronate, and that the contrasting effects of different bisphosphonates on the release of cytokines could be mediated partly through changes in the DNA binding activity of NF-kappaB.

The use of role plays in teaching drug and alcohol management.

This is an evaluation of the use of role plays to teach drug and alcohol assessment and management skills to medical students as part of their general practice training. Role plays of 'real life' scenarios enable students to evaluate their own learning experiences. This method facilitates effective peer discussion of clinical and ethical issues with a high degree of acceptability to both students and teachers.

Is it MS? Presenting symptoms and diagnosis of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is the most common chronic neurological disease in our community. OBJECTIVE: To discuss the presenting symptoms that suggest the diagnosis of MS and consider how to confirm the diagnosis and evaluate the differential diagnosis. DISCUSSION: There are protean presenting symptoms of MS but because of the distribution of pathology predominantly in the peri-ventricular region of the brain, certain symptom complexes are more common. With the recent availability of treatment for MS which has been demonstrated to slow the progress of the disease, the need for early diagnosis has become even more important.

The use and perceived value of diabetes clinical management guidelines in general practice.

OBJECTIVE: To assess attitudes to and use of NSW Health Department (NSWHD) guidelines for the clinical management of diabetes mellitus by divisions of general practice, general practitioners, patients and allied health professionals. METHOD: A mail cross sectional survey of divisions of general practice and a series of focus groups of GPs, patients and allied health professionals, on extent of use and perceived usefulness of the NSWHD guidelines. RESULTS: Forty divisions had or recently concluded a diabetes shared care project. Thirteen out of the 31 diabetes projects with guidelines reported using the NSWHD guidelines. Nineteen divisions were aware of the NSWHD guidelines. While the general response to the guidelines in both the survey and the focus groups was positive, there were suggestions for modifications. Many respondents were not aware of the guidelines. CONCLUSION: Optimal use of the guidelines in general practice requires improved dissemination and an implementation strategy based not only on GP education but also systems to reduce barriers to implementation and to support better quality of GP care.

Impaired blood pressure variability in chronic fatigue syndrome--a potential biomarker.

INTRODUCTION: Autonomic dysfunction is common in chronic fatigue syndrome (CFS). This study set out to derive an autonomic biomarker using a comprehensive assessment of heart rate and blood pressure variability. METHODS: Heart rate and non-invasive continuous blood pressure measurements (task force monitor) at rest and on standing were performed in CFS (Fukuda n = 68) and matched controls (n = 68) to derive high frequency (HF; parasympathetic) and low frequency (LF; sympathetic) heart rate variability (HRV), systolic (SBPV) and diastolic (DBPV) blood pressure variability. Variables of significance were combined using receiver operator curves to explore the diagnostic utility of parameters particularly at rest. RESULTS: At rest, LF-HRV (sympathetic) was significantly increased in CFS compared to controls, while parasympathetic markers were significantly reduced (P = 0.006). Total DBP spectral power was increased (P = 0.0003) across all domains, with a shift towards sympathetic and away from parasympathetic SBPV (P = 0.05). On standing, overall SBPV response was significantly reduced with reductions in both sympathetic and parasympathetic components of SBPV (all P < 0.0001). Change in LF-DBP and relative balance of LF/HF DBP on standing differed between CFS and controls (P < 0.0001). Using the 85% sensitivity levels, we determined a threshold for three chosen resting BPV parameters of LF DBP >3.185, rest HF DBP >0.86, rest total DBP >7.05. Achieving all of these differentiated between CFS and controls with 77% sensitivity and 53% specificity. CONCLUSION: This study has shown that there are objectively measured abnormalities of blood pressure variability in CFS and that these abnormalities have the potential to be a bedside diagnostic tool.

Primary biliary cirrhosis is associated with falls and significant fall related injury.

BACKGROUND: Osteoporosis and autonomic dysfunction are prevalent in the autoimmune liver disease primary biliary cirrhosis (PBC). Postural hypotension is one consequence of autonomic dysfunction and is a recognized risk factor for falls, which, alongside osteoporosis could lead to significant injury and fractures. AIM: To determine the prevalence and sequelae of falls in PBC and to identify modifiable risk factors. DESIGN: Cross-sectional, geographical, population census of PBC and two control groups: primary sclerosing cholangitis and a community dwelling population. Multidisciplinary falls assessment of a representative group of PBC. METHODS: Symptom assessment tools, completed by the three cohorts, determined the prevalence of falls, injuries and associated symptoms. Multidisciplinary assessments, adhering to NICE guidelines, identified modifiable fall associations. RESULTS: Significantly more of the PBC population had fallen (72% P < 0.001) than both control groups. Fifty-five percent had fallen in the last year (P < 0.001), and 22% more than once in the last year (P < 0.01). Seventy percent of PBC fallers were injured, 27% fractured a bone and 19% were admitted to hospital, all significantly more common than controls. Postural dizziness was significantly worse in fallers (P < 0.001), as were balance (P < 0.001) and lower limb strength (P = 0.002). Lower limb strength was independently associated with number of falls in previous year (beta = 0.184, P < 0.001). CONCLUSION: Falls and resultant injury are prevalent in PBC and more common than previously recognized. Addressing postural dizziness, poor balance and lower limb weakness using a multidisciplinary approach has the potential to reduce falls, morbidity and mortality and as a result improve quality of life.

History of multiple sclerosis. An Australian perspective.

In 1868 Jean-Martin Charcot's clinicopathological correlation of multiple sclerosis (MS) was published. Review of Australian medical journals revealed that, 7 years later, Dr Alfred K. Newman, a young physician returning from Aberdeen University, made first mention of MS in the Australian literature when he gave a dissertation entitled 'On insular sclerosis of the brain and spinal cord' which was based on Charcot's teachings. In 1886 Dr Jamieson presented the first case in Australia which fulfilled the criteria for clinically definite MS. This was followed by several other interesting case presentations of MS but it was not until 1915 that Drs Froude Flashman and Oliver Latham presented the first Australian clinicopathological correlation of this disease. Case presentations and extensive discussions regarding the cause of MS appeared in Australian medical journals over the ensuing years but no original Australian research was presented until 1933 when Dr Royle's work on division of the thoracic sympathetic trunk was published. Since that time Australians have made an ever-increasing contribution to the world literature regarding MS pathogenesis, epidemiology and treatment.