A feasibility study using motivational interviewing and a smartphone application to promote physical activity (+Stay-Active) for women with gestational diabetes.

BACKGROUND: Physical activity (PA) interventions have an encouraging role in gestational diabetes mellitus (GDM) management. Digital technologies can potentially be used at scale to support PA. The aim of this study was to assess the feasibility and acceptability of + Stay-Active: a complex intervention which combines motivational interviewing with a smartphone application to promote PA levels in women with GDM. METHODS: This non-randomised feasibility study used a mixed methods approach. Participants were recruited from the GDM antenatal clinic at Oxford University Hospitals. Following baseline assessments (visit 1) including self-reported and device determined PA measurements (wrist worn accelerometer), women participated in an online motivational interview, and then downloaded (visit 2) and used the Stay-Active app (Android or iOS). Women had access to Stay-Active until 36 weeks' gestation, when acceptability and PA levels were reassessed (visit 3). The primary outcome measures were recruitment and retention rates, participant engagement, and acceptability and fidelity of the intervention. Secondary outcome measures included PA levels, app usage, blood glucose and perinatal outcomes. Descriptive statistics were performed for assessments at study visits. Statistics software package Stata 14 and R were used. RESULTS: Over the recruitment period (46 weeks), 114 of 285 women met inclusion criteria and 67 (58%) enrolled in the study. Mean recruitment rate of 1.5 participants/clinic with 2.5 women/clinic meeting inclusion criteria. Fifty-six (83%) received the intervention at visit 2 and 53 (79%) completed the study. Compliance to accelerometer measurement protocols were sufficient in 78% of participants (52/67); wearing the device for more than 10 h on 5 or more days at baseline and 61% (41/67) at 36 weeks. There was high engagement with Stay-Active; 82% (55/67) of participants set goals on Stay-Active. Sustained engagement was evident, participants regularly accessed and logged multiples activities on Stay-Active. The intervention was deemed acceptable; 85% of women rated their care was satisfactory or above, supported by written feedback. CONCLUSIONS: This combined intervention was feasible and accepted. Recruitment rates were lower than expected. However, retention rates remained satisfactory and participant compliance with PA measurements and engagement was a high. Future work will explore the intervention's efficacy to increase PA and impact on clinical outcomes. TRIAL REGISTRATION: The study has received a favourable opinion from South Central-Hampshire B Research Ethics Committee; REC reference: 20/SC/0342. ISRCTN11366562.

Collaboration across the primary/specialist interface in early intervention in psychosis services: a qualitative study.

Background People with new psychotic symptoms may be managed within an Early Intervention in Psychosis service (EIP). They may be discharged back to primary care at the end of their time in an EIP service. Aim To explore the role of primary care in supporting people with psychosis in an EIP service. Design and Setting Qualitative study, within a programme of work to explore the optimum duration of management within an EIP service. Methods Semi-structured interviews with people in EIP services, carers, general practitioners (GPs) and EIP practitioners. Findings GPs report difficulties in referring people into EIP services, have little contact with people who are supported by EIP services and are not included in planning discharge from EIP service to primary care. Conclusions This study suggests that GPs should have a role in the support of people within EIP services (in particular monitoring and managing physical health) and their carers. Inclusion of GPs in managing discharge from EIP services is vital. We suggest that a joint consultation with the service user, their carer (if they wish) along with EIP care co-ordinator and GP would make this transition smoother.

Cardiac Remodeling After Hypertensive Pregnancy Following Physician-Optimized Blood Pressure Self-Management: The POP-HT Randomized Clinical Trial Imaging Substudy.

BACKGROUND: Hypertensive pregnancy disorders are associated with adverse cardiac remodeling, which can fail to reverse in the postpartum period in some women. The Physician-Optimized Postpartum Hypertension Treatment trial demonstrated that improved blood pressure control while the cardiovascular system recovers postpartum associates with persistently reduced blood pressure. We now report the effect on cardiac remodeling. METHODS: In this prospective, randomized, open-label, blinded end point trial, in a single UK hospital, 220 women were randomly assigned 1:1 to self-monitoring with research physician-optimized antihypertensive titration or usual postnatal care from a primary care physician and midwife. Participants were 18 years of age or older, with preeclampsia or gestational hypertension, requiring antihypertensives on hospital discharge postnatally. Prespecified secondary cardiac imaging outcomes were recorded by echocardiography around delivery, and again at blood pressure primary outcome assessment, around 9 months postpartum, when cardiovascular magnetic resonance was also performed. RESULTS: A total of 187 women (101 intervention; 86 usual care) underwent echocardiography at baseline and follow-up, at a mean 258±14.6 days postpartum, of which 174 (93 intervention; 81 usual care) also had cardiovascular magnetic resonance at follow-up. Relative wall thickness by echocardiography was 0.06 (95% CI, 0.07-0.05; P<0.001) lower in the intervention group between baseline and follow-up, and cardiovascular magnetic resonance at follow-up demonstrated a lower left ventricular mass (-6.37 g/m2; 95% CI, -7.99 to -4.74; P<0.001), end-diastolic volume (-3.87 mL/m2; 95% CI, -6.77 to -0.98; P=0.009), and end-systolic volume (-3.25 mL/m2; 95% CI, 4.87 to -1.63; P<0.001) and higher left and right ventricular ejection fraction by 2.6% (95% CI, 1.3-3.9; P<0.001) and 2.8% (95% CI, 1.4-4.1; P<0.001), respectively. Echocardiography-assessed left ventricular diastolic function demonstrated a mean difference in average E/E' of 0.52 (95% CI, -0.97 to -0.07; P=0.024) and a reduction in left atrial volumes of -4.33 mL/m2 (95% CI, -5.52 to -3.21; P<0.001) between baseline and follow-up when adjusted for baseline differences in measures. CONCLUSIONS: Short-term postnatal optimization of blood pressure control after hypertensive pregnancy, through self-monitoring and physician-guided antihypertensive titration, associates with long-term changes in cardiovascular structure and function, in a pattern associated with more favorable cardiovascular outcomes. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04273854.

Long-Term Blood Pressure Control After Hypertensive Pregnancy Following Physician-Optimized Self-Management: The POP-HT Randomized Clinical Trial.

Importance: Pregnancy hypertension results in adverse cardiac remodeling and higher incidence of hypertension and cardiovascular diseases in later life. Objective: To evaluate whether an intervention designed to achieve better blood pressure control in the postnatal period is associated with lower blood pressure than usual outpatient care during the first 9 months postpartum. Design, Setting, and Participants: Randomized, open-label, blinded, end point trial set in a single hospital in the UK. Eligible participants were aged 18 years or older, following pregnancy complicated by preeclampsia or gestational hypertension, requiring antihypertensive medication postnatally when discharged. The first enrollment occurred on February 21, 2020, and the last follow-up, November 2, 2021. The follow-up period was approximately 9 months. Interventions: Participants were randomly assigned 1:1 to self-monitoring along with physician-optimized antihypertensive titration or usual postnatal care. Main Outcomes and Measures: The primary outcome was 24-hour mean diastolic blood pressure at 9 months postpartum, adjusted for baseline postnatal blood pressure. Results: Two hundred twenty participants were randomly assigned to either the intervention group (n = 112) or the control group (n = 108). The mean (SD) age of participants was 32.6 (5.0) years, 40% had gestational hypertension, and 60% had preeclampsia. Two hundred participants (91%) were included in the primary analysis. The 24-hour mean (SD) diastolic blood pressure, measured at 249 (16) days postpartum, was 5.8 mm Hg lower in the intervention group (71.2 [5.6] mm Hg) than in the control group (76.6 [5.7] mm Hg). The between-group difference was -5.80 mm Hg (95% CI, -7.40 to -4.20; P < .001). Similarly, the 24-hour mean (SD) systolic blood pressure was 6.5 mm Hg lower in the intervention group (114.0 [7.7] mm Hg) than in the control group (120.3 [9.1] mm Hg). The between-group difference was -6.51 mm Hg (95% CI, -8.80 to -4.22; P < .001). Conclusions and Relevance: In this single-center trial, self-monitoring and physician-guided titration of antihypertensive medications was associated with lower blood pressure during the first 9 months postpartum than usual postnatal outpatient care in the UK. Trial Registration: ClinicalTrials.gov Identifier: NCT04273854.

Characterisation of the treatment provided for children with unilateral hearing loss.

Background: Children with permanent unilateral hearing loss (UHL) are an understudied population, with limited data to inform the guidelines on clinical management. There is a funding gap in healthcare provision for the children with UHL in the United Kingdom, where genetic screening, support services, and devices are not consistently provided or fully funded in all areas. They are a disparate population with regard to aetiology and their degree of hearing loss, and hence their device choice and use. Despite having one "good ear", some children with UHL can have similar outcomes, socially, behaviourally, and academically, to children with bilateral hearing loss, highlighting the importance of understanding this population. In this longitudinal cohort study, we aimed to characterise the management of the children with UHL and the gaps in the support services that are provided for the children in Nottingham, United Kingdom. Methods: A cohort study was conducted collecting longitudinal data over 17 years (2002-2019) for 63 children with permanent congenital confirmed UHL in a large tertiary regional referral centre for hearing loss in Nottingham, United Kingdom. The cases of UHL include permanent congenital, conductive, mixed, or sensorineural hearing loss, and the degree of hearing loss ranges from mild to profound. The data were taken from their diagnostic auditory brainstem responses and their two most recent hearing assessments. Descriptors were recorded of the devices trialled and used and the diagnoses including aetiology of UHL, age of first fit, degree of hearing loss, when and which type of device was used, why a device was not used, the support services provided, concerns raised, and who raised them. Results: Most children (45/63; 71%) trialled a device, and the remaining 18 children had no device trial on record. Most children (20/45; 44%) trialled a bone-conduction device, followed by contralateral routing of signal aid (15/45; 33%) and conventional hearing aids (9/45; 20%). Most children (36/45; 80%) who had a device indicated that they wore their device "all day" or every day in school. Few children (8/45; 18%) reported that they wore their device rarely, and the reasons for this included bullying (3/8), feedback from the device (2/8), and discomfort from the device (2/8). Only one child reported that the device was not helping with their hearing. The age that the children were first fitted with their hearing device varied a median of 2.5 years for hearing aids and bone-conduction devices and 7 years for a contralateral routing of signal aid. The length of time that the children had the device also varied widely (median of 26 months, range 3-135 months); the children had their bone-conduction hearing aid for the longest period of time (median of 32.5 months). There was a significant trend where more recent device fittings were happening for children at a younger age. Fifty-one children were referred by the paediatric audiologist to a support service, 72.5% (37/51) were subsequently followed up by the referred service with no issue, whilst the remaining 27.5% (14/51) encountered an issue leading to an unsuccessful provision of support. Overall, most children (65%, 41/63) had no reported concerns, and 28.5% (18/63) of the children went on to have a documented concern at some point during their audiological care: five with hearing aid difficulties, five with speech issues, four with no improvement in hearing, three facing self-image or bullying issues, and one case of a child struggling to interact socially with friends. Three of these children had not trialled a device. We documented every concern reported from the parents, clinicians, teachers of the deaf, and from the children themselves. Where concerns were raised, more than half (58.6%, 10/18) were by schools and teachers, the remaining four concerns were raised by the family, and further four concerns were raised by the children themselves. Conclusion: To discover what management will most benefit which children with permanent UHL, we first must characterise their treatment, their concerns, and the support services available for them. Despite the children with UHL being a highly disparate population-in terms of their aetiology, their device use, the degree of hearing loss, and the age at which they trial a device-the majority report they use their device mostly in school. In lieu of available data and in consideration of the devices that are available to them, it could be useful to support families and clinicians in understanding the devices which are most used and where they are used. Considering the reasons for cessation of regular device use counselling and support services would be vital to support the children with UHL.

Effects of environmental impact and nutrition labelling on food purchasing: An experimental online supermarket study.

Nutrition labels and ecolabels can support consumers to make healthier and more sustainable choices, and the former is now widespread. But there is no information on the impact of ecolabels in the presence of nutrition labels. The aims of this study were primarily to examine whether (1) ecolabels are effective at promoting sustainable purchasing behaviour if presented alongside nutrition labels; (2) and secondarily, whether nutrition labels are effective at promoting healthier purchasing if presented alongside ecolabels. Participants (N = 2730) visited an experimental online supermarket platform, and were randomised to see products with (1) environmental impact labels only; (2) nutrition (NutriScore) labels only; (3) both environmental and nutrition labels; (4) no labels. Linear regressions compared the mean environmental impact scores (EIS; primary outcome) and health scores of products in participants' shopping baskets across each condition. Compared to control (no labels) there were significant reductions in the EIS when environmental impact labels were presented: Alone (-1.3, 95%CI: -2.3 to -0.4) or With nutrition labels (-2.0, 95%CI: -2.9 to -1.0), with no evidence of differences in effectiveness between these two conditions. There was no evidence of an impact of nutrition labels on either the EIS or the healthiness of purchases, both when nutrition labels were shown alone and when ecolabels were also present. Environmental impact labels may be effective at encouraging more sustainable purchases alone or when used alongside nutrition labels. This adds to the evidence base on the feasibility and effectiveness of environmental impact labelling as an important measure to change dietary behaviour to improve planetary health.

Effects of environmental impact labels on the sustainability of food purchases: Two randomised controlled trials in an experimental online supermarket.

Providing consumers with product-specific environmental impact information for food products (ecolabels) may promote more sustainable purchasing, needed to meet global environmental targets. Two UK studies investigated the effectiveness of different ecolabels using an experimental online supermarket platform. Study 1 (N = 1051 participants) compared three labels against control (no label), while Study 2 (N = 4979) tested four designs against control. Study 1 found significant reductions in the environmental impact score (EIS) for all labels compared to control (labels presented: values for four environmental indicators [-3.9 percentiles, 95%CIs: -5.2,-2.6]; a composite score [taking values from A to E; -3.9, 95%CIs: -5.2,-2.5]; or both together [-3.2, 95%CIs: -4.5,-1.9]). Study 2 showed significant reductions in EIS compared to control for A-E labels [-2.3, 95%CIs: -3.0,-1.5], coloured globes with A-E scores [-3.2, 95%CIs:-3.9,-2.4], and red globes highlighting 'worse' products [-3.2, 95%CIs:-3.9,-2.5]. There was no evidence that green globes highlighting 'better' products were effective [-0.5, 95%CIs:-1.3,0.2]. Providing ecolabels is a promising intervention to promote the selection of more sustainable products.

Study protocol: use of a smartphone application to support the implementation of a complex physical activity intervention (+Stay Active) in women with gestational diabetes mellitus-protocol for a non-randomised feasibility study.

INTRODUCTION: Physical activity (PA) interventions have a promising role in the management of gestational diabetes mellitus (GDM). Digital technologies can support PA at scale and remotely. The protocol describes a study designed to determine the feasibility and acceptability of a complex intervention; known as +Stay Active. +Stay Active combines motivational interviewing with a bespoke behaviour change informed smartphone application (Stay-Active) to augment PA levels in women with GDM. METHODS AND ANALYSIS: This is a non-randomised feasibility study using a mixed methods approach. Participants will be recruited from the GDM antenatal clinic at the Women Centre, John Radcliffe Hospital, Oxford. Following baseline assessments (visit 1) including self-reported and device determined PA assessment (wearing a wrist accelerometer), women will be invited to participate in an online motivational interview, then download and use the Stay-Active app (Android or iOS) (visit 2). Women will have access to Stay-Active until 36 weeks gestation, when engagement and PA levels will be reassessed (visit 3). The target sample size is 60 women. Primary outcomes are recruitment and retention rates, compliance and assessment of participant engagement and acceptability with the intervention. Secondary outcomes are assessment of blood glucose control, self-reported and device determined assessment of PA, usage and structured feedback of participant's attitudes to +Stay Active, assessment of health costs and description of maternal and neonatal outcomes. This study will provide key insights into this complex intervention regarding engagement in smartphone technology and the wearing of accelerometers. These data will inform the development of a randomised controlled trial with refinements to intervention implementation. ETHICS AND DISSEMINATION: The study has received a favourable opinion from South Central-Hampshire B Research Ethics Committee; REC reference: 20/SC/0342. Written informed consent will be obtained from all participants. Findings will be disseminated through peer-reviewed journals, conferences and seminar presentations. TRIAL REGISTRATION NUMBER: ISRCTN11366562.

Oxford Food and Activity Behaviors 20-item questionnaire to assess personal weight management strategies: Development and testing.

OBJECTIVE: The aim of this study was to develop a shortened Oxford Food and Activity Behaviors (OxFAB) questionnaire to identify the cognitive and behavioral strategies used by individuals during weight-management attempts. METHODS: This study reduced an existing 117-item questionnaire (the original OxFAB questionnaire) through identifying clusters of techniques from the responses of 278 people living with obesity and, within those clusters, identifying the most representative question or questions. Questions were rephrased to cover multiple strategies at the domain level, with several alternative phrasings developed for new questions. Face validity was tested through think-aloud interviews with 12 people living with obesity. Questions were rephrased accordingly and tested using test-retest (n = 172). Prevalence- and bias-adjusted κ (PABAK) were calculated, and questions with PABAK < 0.41 were rewritten and evaluated in a new test-retest sample (n = 130). RESULTS: OxFAB20 consists of 20 questions covering diet, physical activity, and cognitive strategies for weight management. Test-retest resulted in a mean PABAK score of 0.56 (SD = 0.14). Questions were revised where appropriate. The questionnaire is available for use via a CC-BY license. CONCLUSIONS: The OxFAB20 questionnaire provides a practical tool for researchers to identify the cognitive and behavioral strategies used by individuals during attempts at weight control.

Impact of increasing the relative availability of meat-free options on food selection: two natural field experiments and an online randomised trial.

BACKGROUND: Increasing the availability of lower energy-density foods is a promising intervention to encourage healthier food purchasing but few studies have examined the effect of increasing availability of meat-free meals to promote more sustainable purchasing. We report three studies, all examining the impact of altering the availability of meat-free meals on meal selection. METHODS: Study 1 (a natural experiment in one university cafeteria) examined the impact of altering the ratio of meat-free meals (one meat-free and two meat, to two meat-free and one meat) on weekly sales of meals containing meat. Study 2 (a natural experiment in 18 worksite cafeterias) examined the impact on meat-free meal sales of a menu change designed to increase the availability of meat-free meals. Study 3 (an online study of 2205 UK-representative adults) compared meal selections when participants were randomised to ranges comprised of (a) one meat-free, three meat options; (b) two meat-free, two meat; or (c) three meat-free, one meat. RESULTS: Study 1 suggested a significant decrease in the proportion of sales of meat options when the availability of meat-free options increased (- 19.9 percentage points; 95%CIs:-25.2,-14.6), with no evidence of changes to meat-based meal sales in other university cafeterias during the same period. Findings from Study 2 were mixed: multilevel regressions found no evidence of an increase in meat-free meals following the menu change (2.3 percentage points; 95%CIs: - 1.3,5.9), while interrupted time-series analyses suggested sales did increase (2.3; 95%CIs: 0.4,4.2), but implementation of the planned change was limited. In Study 3 reducing meat-free options from 50 to 25% reduced participants' selection of meat-free options (odds ratio 0.35; 95%CIs: 0.26,0.46), while increasing meat-free options from 50 to 75% increased meat-free selections (odds ratio 2.43; 95%CIs: 1.94,3.04). There was no evidence effects were moderated by gender, socioeconomic status or usual meat consumption. CONCLUSION: Increasing the availability of meat-free options is effective at reducing meat selection and purchasing for different ratios of meat to meat-free options. The magnitude of the effect is uncertain, but with no evidence of differences in response by demographic groups when directly tested. TRIAL REGISTRATION: Study 3: Open Science Framework; https://osf.io/ze9c6 ; 6/8/2020.

Donor age significantly influences the Raman spectroscopic biomolecular fingerprint of human pancreatic extracellular matrix proteins following collagenase-based digestion.

Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI). However, age-related differences in pancreatic matrix digestion have not been studied in detail at the molecular level. To address this, we investigated ECM digestion in purified ECM proteins and in pancreatic tissue sections from younger (≤30 years; n = 5) and older (>55 years; n = 5) BMI matched donors, using Raman microspectroscopy (RMS). The Raman spectral profiles for purified collagens I, IV, VI and laminins were significantly altered following controlled enzyme treatment. Pancreatic cryosections were treated with Serva collagenase, NP, or the two enzymes combined, at clinically relevant concentrations. RMS demonstrated that the ECM at the islet-exocrine interface was differentially digested with respect to donor age. The action of collagenase was affected to a greater extent than NP. RMS is a powerful, marker-independent technology for characterising the human pancreatic ECM and demonstrating differences between donor types. Ongoing detailed studies using RMS will assist the development of donor-specific enzyme blends, increasing the overall success of human islet isolation and benefiting many people with type 1 diabetes worldwide. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally invasive treatment, which can reverse Type 1 Diabetes Mellitus (T1DM) in selected patients. Islets of Langerhans are extracted (isolated) from the exocrine tissue of human donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current enzymes are ineffective at digesting pancreases from younger donors. Using Raman microspectroscopy we demonstrate that donor age affects the enzymatic digestion of the pancreatic ECM at the molecular level. Collagenase activity is affected to a greater extent than NP. These findings will assist the development of donor-specific enzymes, thereby increasing the overall success of islet isolation and benefiting many people with T1DM worldwide.

Does Islet Size Really Influence Graft Function After Clinical Islet Transplantation?

BACKGROUND: It has been proposed that islet transplants comprised primarily of small rather than large islets may provide better graft function, due to their lower susceptibility to hypoxic damage. Our aim was to determine whether islet size correlated with in vivo graft function in islet transplant recipients with C peptide-negative type 1 diabetes when islets have undergone pretransplant islet culture. METHODS: Human pancreatic islets were isolated, cultured for 24 hours and infused by standardized protocols. Ninety-minute stimulated C-peptide concentrations were determined during a standard meal tolerance test 3 months posttransplant. The islet isolation index (IEq/islet number) was determined immediately after isolation and again before transplantation (after tissue culture). This was correlated with patient insulin requirement or stimulated C-peptide. RESULTS: Changes in insulin requirement did not significantly correlate with islet isolation index. Stimulated C-peptide correlated weakly with IEq at isolation (P = 0.40) and significantly with IEq at transplantation (P = 0.018). Stimulated C-peptide correlated with islet number at isolation (P = 0.013) and more strongly with the islet number at transplantation (P = 0.001). In contrast, the correlation of stimulated C-peptide and islet isolation index was weaker (P = 0.018), and this was poorer at transplantation (P = 0.034). Using linear regression, the strongest association with graft function was islet number (r = 0.722, P = 0.001). Islet size was not related to graft function after adjusting for islet volume or number. CONCLUSIONS: These data show no clear correlation between islet isolation index and graft function; both small and large islets are suitable for transplantation, provided the islets have survived a short culture period postisolation.

Development of a Simple In Vitro Assay to Assess Digestion of the Extracellular Matrix of the Human Pancreas by Collagenase Enzyme Blends.

Despite huge advances in the field of islet transplantation over the last two decades, current islet isolation methods remain suboptimal, with transplantable yields obtained in less than half of all pancreases processed worldwide. Successful islet isolation is dependent on the ability of collagenase-based enzyme blends to digest extracellular matrix components at the islet-exocrine interface. The limited availability of donor pancreases hinders the use of full-scale islet isolations to characterize pancreas digestion by different enzyme components or blends, or allow the influence of inter-pancreatic variability between donors to be explored. We have developed a method that allows multiple enzyme components to be tested on any one pancreas. Biopsies of 0.5 cm3 were taken from seven standard (age ≥45) and eight young (age ≤35) pancreases. Serial cryosections were treated with Serva collagenase, neutral protease (NP), or the two enzymes together at clinically relevant concentrations. Following digestion, insulin and either collagen IV or laminin-α5 were detected by immunofluorescent labeling. Protein loss at the islet-exocrine interface was semi-quantified morphometrically, with reference to a control section. Differential digestion of the two proteins based on the enzyme components used was seen, with protein digestion significantly influenced by donor age. Treatment with collagenase and NP alone was significantly more effective at digesting collagen IV in the standard donor group, as was the NP mediated digestion of laminin-α5. Collagenase alone was not capable of significantly digesting laminin-α5 in either donor group. Combining the two enzymes ameliorated the age-related differences in the digestion of both proteins. No significant differences in protein loss were detected by the method when analyzed by two independent operators, demonstrating the reproducibility of the assay. The development of this simple yet reproducible assay has implications for both enzyme batch testing and identifying inter-donor digestion variability, while utilizing small amounts of both enzyme and human tissue.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess ß-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet ß-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Impact of saturated, polyunsaturated and monounsaturated fatty acid-rich micelles on lipoprotein synthesis and secretion in Caco-2 cells.

Meal fatty acids have been shown to modulate the size and composition of triacylglycerol (TAG)-rich lipoproteins influencing the magnitude and duration of the postprandial plasma TAG response. As a result there is considerable interest in the origin of these meal fatty-acid induced differences in particle composition. Caco-2 cells were incubated over 4 days with fatty acid mixtures resembling the composition of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA)-rich meals fed in a previous postprandial study to determine their impact on lipoprotein synthesis and secretion. The MUFA- and PUFA-rich mixtures supported greater intracellular TAG, but not cholesterol accumulation compared with the SFA-rich mixture (P < 0.001). The MUFA-rich mixture promoted significantly greater TAG and cholesterol secretion than the other mixtures and significantly more apolipoprotein B-100 secretion than the PUFA-rich mixture (P < 0.05). Electron microscopy revealed the SFA-rich mixture had led to unfavourable effects on cellular morphology, compared with the unsaturated fatty acid-rich mixtures. Our findings suggest the MUFA-rich mixture, may support the formation of a greater number of TAG-rich lipoproteins, which is consistent with indirect observations from our human study. Our electron micrographs are suggestive that some endocytotic uptake of MUFA-rich taurocholate micelles may promote greater lipoprotein synthesis and secretion in Caco-2 cells.

Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids.

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.

Apolipoprotein E enrichment of immuno-separated chylomicron and chylomicron remnants following saturated fatty acids.

AIM: We examined the effect of meal fatty acids on lipid and apolipoprotein concentrations of very low density lipoprotein (VLDL) and chylomicron/chylomicron remnants in lipid fractions with a Svedberg flotation rate (Sf) 60-400 and Sf 20-60. METHODS AND RESULTS: Six healthy middle-aged men received in random order mixed meals enriched with saturated (SFA), polyunsaturated (PUFA) or monounsaturated (MUFA) fatty acids on 3 occasions. VLDL and chylomicron/chylomicron remnants in the lipid fractions were separated by immunoaffinity chromatography against apo B-100. In the Sf 60-400 chylomicron/chylomicron remnants, triacylglycerol and cholesterol concentrations were significantly lower following PUFA compared with SFA and MUFA (P < or = 0.05). Apolipoprotein (apo) E responses were significantly higher after SFA in chylomicron/chylomicron remnants and VLDL compared with PUFA and MUFA (P < 0.007). However, apo B responses (particle number) were higher following MUFA than SFA (P = 0.039 for chylomicron/chylomicron remnants). Composition of the chylomicron/chylomicron remnants (expressed per particle) revealed differences in their triacylglycerol and apo E contents; in the Sf 60-400 fraction, SFA-rich chylomicron/chylomicron remnants contained significantly more triacylglycerol than MUFA (P = 0.028), more apo E than PUFA- and MUFA-rich particles (P < 0.05) and in the Sf 20-60 fraction, more apo E than MUFA (P = 0.009). CONCLUSION: There are specific differences in the composition of chylomicron/chylomicron remnants formed after saturated compared with unsaturated fatty acid-rich meals which could determine their metabolic fate in the circulation and subsequent atherogenicity.

Acute effects of meal fatty acids on postprandial NEFA, glucose and apo E response: implications for insulin sensitivity and lipoprotein regulation?

Our aim was to determine whether meal fatty acids influence insulin and glucose responses to mixed meals and whether these effects can be explained by variations in postprandial NEFA and Apo, which regulate the metabolism of triacylglycerol-rich lipoproteins (Apo C and E). A single-blind crossover study examined the effects of single meals enriched in saturated fatty acids SFA), n-6 PUFA and MUFA on plasma metabolite and insulin responses. The triacylglycerol response following the PUFA meal showed a lower net incremental area under the curve than following the SFA and MUFA meals (P<0.007). Compared with the SFA meal, the PUFA meal showed a lower net incremental area under the curve for the NEFA response from initial suppression to the end of the postprandial period (180-480 min; P<0.02), and both PUFA and MUFA showed a lower net incremental glucose response (P<0.02), although insulin concentrations were similar between meals. The pattern of the Apo E response was also different following the SFA meal (P<0.02). There was a significant association between the net incremental NEFA (180-480 min) and glucose response (rs=0.409, P=0.025), and in multiple regression analysis the NEFA response accounted for 24 % of the variation in glucose response. Meal SFA have adverse effects on the postprandial glucose response that may be due to greater elevations in NEFA arising from differences in the metabolism of SFA- v. PUFA- and MUFA-rich lipoproteins. Elevated Apo E responses to high-SFA meals may have important implications for the hepatic metabolism of triacylglycerol-rich lipoproteins.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages.

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subfractions and the relative atherogenicity of these subfractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation (S(f)) rates: chylomicron (CM, S(f)>400), very low-density lipoprotein (VLDL)-1 (S(f) 60-400) and VLDL-2 (S(f) 20-60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-1 caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles.