59,856 research outputs found

    Baculovirus-Mediated Expression of Human 65 kDa and 67 kDa Glutamic Acid Decarboxylases in SF9 Insect Cells and Their Relevance in Diagnosis of Insulin-Dependent Diabetes Mellitus

    Get PDF
    cDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GAD65 and GAD67) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD65 and GAD67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD65 and GAD67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD65 and GAD67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed

    A randomised controlled study of high intensity exercise as a dishabituating stimulus to improve hypoglycaemia awareness in people with type 1 diabetes:a proof of concept study

    Get PDF
    Aims/hypothesis Approximately 25% of people with type 1 diabetes have suppressed counterregulatory hormonal and symptomatic responses to insulin-induced hypoglycaemia, which renders them at increased risk of severe, disabling hypoglycaemia. This is called impaired awareness of hypoglycaemia (IAH), the cause of which is unknown. We recently proposed that IAH develops through habituation, a form of adaptive memory to preceding hypoglycaemia. Consistent with this hypothesis, we demonstrated restoration of defective counterregulatory hormonal responses to hypoglycaemia (referred to as dishabituation) in a rodent model of IAH following introduction of a novel stress stimulus (high intensity training [HIT]). In this proof-of-concept study we sought to further test this hypothesis by examining whether a single episode of HIT would amplify counterregulatory responses to subsequent hypoglycaemia in people with type 1 diabetes who had IAH (assessed by Gold score ≥4, modified Clarke score ≥4 or Dose Adjustment For Normal Eating (DAFNE) hypoglycaemia awareness rating 2 or 3). The primary outcome was the difference in adrenaline response to hypoglycaemia following both a single episode of HIT and rest. Methods In this randomised, crossover study 12 participants aged between 18 and 55 years with type 1 diabetes for ≥5 years and an HbA1c < 75 mmol/mol (9%) were recruited. Individuals were randomised using computer generated block randomisation to start with one episode of HIT (4 × 30 s cycle sprints [2 min recovery] at 150% of maximum wattage achieved during V˙O2peak assessment) or rest (control). The following day they underwent a 90 min hyperinsulinaemic–hypoglycaemic clamp study at 2.5 mmol/l with measurement of hormonal counterregulatory response, symptom scores and cognitive testing (four-choice reaction time and digit symbol substitution test). Each intervention and subsequent clamp study was separated by at least 2 weeks. The participants and investigators were not blinded to the intervention or measurements during the study. The investigators were blinded to the primary outcome and blood analysis results. Results All participants (six male and six female, age 19–54 years, median [IQR] duration of type 1 diabetes 24.5 [17.3–29.0] years, mean [SEM] HbA1c 56 [3.67] mmol/mol; 7.3% [0.34%]) completed the study (both interventions and two clamps). In comparison with the rest study, a single episode of HIT led to a 29% increase in the adrenaline (epinephrine) response (mean [SEM]) (2286.5 [343.1] vs 2953.8 [384.9] pmol/l); a significant increase in total symptom scores (Edinburgh Hypoglycaemia Symptom Scale: 24.25 [2.960 vs 27.5 [3.9]; p < 0.05), and a significant prolongation of four-choice reaction time (591.8 [22.5] vs 659.9 [39.86] ms; p < 0.01] during equivalent hypoglycaemia induced the following day. Conclusions/interpretation These findings are consistent with the hypothesis that IAH develops in people with type 1 diabetes as a habituated response and that introduction of a novel stressor can restore, at least partially, the adapted counterregulatory hormonal, symptomatic and cognitive responses to hypoglycaemia.Output Status: Forthcoming/Available Onlin

    Randomized multicentre pilot study of sacubitril/valsartan versus irbesartan in patients with chronic kidney disease: United Kingdom Heart and Renal Protection (HARP)- III—rationale, trial design and baseline data

    Get PDF
    BACKGROUND: Patients with chronic kidney disease (CKD) are at risk of progression to end-stage renal disease and cardiovascular disease. Data from other populations and animal experiments suggest that neprilysin inhibition (which augments the natriuretic peptide system) may reduce these risks, but clinical trials among patients with CKD are required to test this hypothesis. METHODS: UK Heart and Renal Protection III (HARP-III) is a multicentre, double-blind, randomized controlled trial comparing sacubitril/valsartan 97/103 mg two times daily (an angiotensin receptor-neprilysin inhibitor) with irbesartan 300 mg one time daily among 414 patients with CKD. Patients ≥18 years of age with an estimated glomerular filtration rate (eGFR) of ≥45 but &lt;60 mL/min/1.73 m2 and urine albumin:creatinine ratio (uACR) &gt;20 mg/mmol or eGFR ≥20 but &lt;45 mL/min/1.73 m2 (regardless of uACR) were invited to be screened. Following a 4- to 7-week pre-randomization single-blind placebo run-in phase (during which any current renin-angiotensin system inhibitors were stopped), willing and eligible participants were randomly assigned either sacubitril/valsartan or irbesartan and followed-up for 12 months. The primary aim was to compare the effects of sacubitril/valsartan and irbesartan on measured GFR after 12 months of therapy. Important secondary outcomes include effects on albuminuria, change in eGFR over time and the safety and tolerability of sacubitril/valsartan in CKD. RESULTS: Between November 2014 and January 2016, 620 patients attended a screening visit and 566 (91%) entered the pre-randomization run-in phase. Of these, 414 (73%) participants were randomized (mean age 63 years; 72% male). The mean eGFR was 34.0 mL/min/1.73 m2 and the median uACR was 58.5 mg/mmol. CONCLUSIONS: UK HARP-III will provide important information on the short-term effects of sacubitril/valsartan on renal function, tolerability and safety among patients with CKD

    Integrated out-of-hours care arrangements in England: observational study of progress towards single call access via NHS Direct and impact on the wider health system

    Get PDF
    Objectives: To assess the extent of service integration achieved within general practice cooperatives and NHS Direct sites participating in the Department of Health’s national “Exemplar Programme” for single call access to out-of-hours care via NHS Direct. To assess the impact of integrated out-of-hours care arrangements upon general practice cooperatives and the wider health system (use of emergency departments, 999 ambulance services, and minor injuries units). Design: Observational before and after study of demand, activity, and trends in the use of other health services. Setting: Thirty four English general practice cooperatives with NHS Direct partners (“exemplars”) of which four acted as “case exemplars”. Also 10 control cooperatives for comparison. Main Outcome Measures: Extent of integration achieved (defined as the proportion of hours and the proportion of general practice patients covered by integrated arrangements), patterns of general practice cooperative demand and activity and trends in use of the wider health system in the first year. Results: Of 31 distinct exemplars 21 (68%) integrated all out-of-hours call management by March 2004. Nine (29%) established single call access for all patients. In the only case exemplar where direct comparison was possible, cooperative nurse telephone triage before integration completed a higher proportion of calls with telephone advice than did NHS Direct afterwards (39% v 30%; p<0.0001). The proportion of calls completed by NHS Direct telephone advice at other sites was lower. There is evidence for transfer of demand from case exemplars to 999 ambulance services. A downturn in overall demand for care seen in two case exemplars was also seen in control sites. Conclusion: The new model of out-of-hours care was implemented in a variety of settings across England by new partnerships between general practice cooperatives and NHS Direct. Single call access was not widely implemented and most patients needed to make at least two telephone calls to contact the service. In the first year, integration may have produced some reduction in total demand, but this may have been accompanied by shifts from one part of the local health system to another. NHS Direct demonstrated capability in handling calls but may not currently have sufficient capacity to support national implementation

    Measuring Blood Glucose Concentrations in Photometric Glucometers Requiring Very Small Sample Volumes

    Full text link
    Glucometers present an important self-monitoring tool for diabetes patients and therefore must exhibit high accu- racy as well as good usability features. Based on an invasive, photometric measurement principle that drastically reduces the volume of the blood sample needed from the patient, we present a framework that is capable of dealing with small blood samples, while maintaining the required accuracy. The framework consists of two major parts: 1) image segmentation; and 2) convergence detection. Step 1) is based on iterative mode-seeking methods to estimate the intensity value of the region of interest. We present several variations of these methods and give theoretical proofs of their convergence. Our approach is able to deal with changes in the number and position of clusters without any prior knowledge. Furthermore, we propose a method based on sparse approximation to decrease the computational load, while maintaining accuracy. Step 2) is achieved by employing temporal tracking and prediction, herewith decreasing the measurement time, and, thus, improving usability. Our framework is validated on several real data sets with different characteristics. We show that we are able to estimate the underlying glucose concentration from much smaller blood samples than is currently state-of-the- art with sufficient accuracy according to the most recent ISO standards and reduce measurement time significantly compared to state-of-the-art methods

    Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes

    Get PDF
    Skeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers. We profiled &gt;47,000 mRNA sequences and &gt;500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes. The muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P &lt; 7.4 × 10-8), post-translational modification (P &lt; 9.7 × 10-5) and developmental (P &lt; 1.3 × 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation. We provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions

    Lower Ankle-Brachial Index Is Related to Worse Cognitive Performance in Old Age

    Get PDF
    Objective: We aimed to study the associations between peripheral artery disease (PAD) and ankle-brachial index (ABI) and performance in a range of cognitive domains in nondemented elderly persons. Methods: Data were collected within the Lothian Birth Cohort 1921 and 1936 studies. These are two narrow-age cohorts at age 87 (n = 170) and 73 (n = 748) years. ABI was analyzed as a dichotomous (PAD vs. no PAD) and a continuous measure. PAD was defined as having an ABI less than 0.90. Measures of nonverbal reasoning, verbal declarative memory, verbal fluency, working memory, and processing speed were administered. Both samples were screened for dementia. Results: We observed no significant differences in cognitive performance between persons with or without PAD. However, higher ABI was associated with better general cognition (β = .23, p = .02, R(2) change = .05) and processing speed (β = .29, p < .01, R(2) change = .08) in the older cohort and better processing speed (β = .12, p < .01, R(2) change = .01) in the younger cohort. This was after controlling for age, sex, and childhood mental ability and excluding persons with abnormally high ABI (>1.40) and a history of cardiovascular or cerebrovascular disease. Conclusion: Lower ABI is associated with worse cognitive performance in old age, especially in the oldest old (>85 years), possibly because of long-term exposure to atherosclerotic disease. Interventions targeting PAD in persons free of manifest cardiovascular and cerebrovascular disease may reduce the incidence of cognitive impairment and dementia
    corecore