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1.  Effects of High-Intensity Interval Exercise versus Moderate Continuous Exercise on Glucose Homeostasis and Hormone Response in Patients with Type 1 Diabetes Mellitus Using Novel Ultra-Long-Acting Insulin 
PLoS ONE  2015;10(8):e0136489.
Introduction
We investigated blood glucose (BG) and hormone response to aerobic high-intensity interval exercise (HIIE) and moderate continuous exercise (CON) matched for mean load and duration in type 1 diabetes mellitus (T1DM).
Material and Methods
Seven trained male subjects with T1DM performed a maximal incremental exercise test and HIIE and CON at 3 different mean intensities below (A) and above (B) the first lactate turn point and below the second lactate turn point (C) on a cycle ergometer. Subjects were adjusted to ultra-long-acting insulin Degludec (Tresiba/ Novo Nordisk, Denmark). Before exercise, standardized meals were administered, and short-acting insulin dose was reduced by 25% (A), 50% (B), and 75% (C) dependent on mean exercise intensity. During exercise, BG, adrenaline, noradrenaline, dopamine, cortisol, glucagon, and insulin-like growth factor-1, blood lactate, heart rate, and gas exchange variables were measured. For 24 h after exercise, interstitial glucose was measured by continuous glucose monitoring system.
Results
BG decrease during HIIE was significantly smaller for B (p = 0.024) and tended to be smaller for A and C compared to CON. No differences were found for post-exercise interstitial glucose, acute hormone response, and carbohydrate utilization between HIIE and CON for A, B, and C. In HIIE, blood lactate for A (p = 0.006) and B (p = 0.004) and respiratory exchange ratio for A (p = 0.003) and B (p = 0.003) were significantly higher compared to CON but not for C.
Conclusion
Hypoglycemia did not occur during or after HIIE and CON when using ultra-long-acting insulin and applying our methodological approach for exercise prescription. HIIE led to a smaller BG decrease compared to CON, although both exercises modes were matched for mean load and duration, even despite markedly higher peak workloads applied in HIIE. Therefore, HIIE and CON could be safely performed in T1DM.
Trial Registration
ClinicalTrials.gov NCT02075567 http://www.clinicaltrials.gov/ct2/show/NCT02075567
doi:10.1371/journal.pone.0136489
PMCID: PMC4552855  PMID: 26317981
2.  Accuracy of Continuous Glucose Monitoring During Three Closed-Loop Home Studies Under Free-Living Conditions 
Diabetes Technology & Therapeutics  2015;17(11):801-807.
Abstract
Objectives: Closed-loop (CL) systems modulate insulin delivery based on glucose levels measured by a continuous glucose monitor (CGM). Accuracy of the CGM affects CL performance and safety. We evaluated the accuracy of the Freestyle Navigator® II CGM (Abbott Diabetes Care, Alameda, CA) during three unsupervised, randomized, open-label, crossover home CL studies.
Materials and Methods: Paired CGM and capillary glucose values (10,597 pairs) were collected from 57 participants with type 1 diabetes (41 adults [mean±SD age, 39±12 years; mean±SD hemoglobin A1c, 7.9±0.8%] recruited at five centers and 16 adolescents [mean±SD age, 15.6±3.6 years; mean±SD hemoglobin A1c, 8.1±0.8%] recruited at two centers). Numerical accuracy was assessed by absolute relative difference (ARD) and International Organization for Standardization (ISO) 15197:2013 15/15% limits, and clinical accuracy was assessed by Clarke error grid analysis.
Results: Total duration of sensor use was 2,002 days (48,052 h). Overall sensor accuracy for the capillary glucose range (1.1–27.8 mmol/L) showed mean±SD and median (interquartile range) ARD of 14.2±15.5% and 10.0% (4.5%, 18.4%), respectively. Lowest mean ARD was observed in the hyperglycemic range (9.8±8.8%). Over 95% of pairs were in combined Clarke error grid Zones A and B (A, 80.1%, B, 16.2%). Overall, 70.0% of the sensor readings satisfied ISO criteria. Mean ARD was consistent (12.3%; 95% of the values fall within ±3.7%) and not different between participants (P=0.06) within the euglycemic and hyperglycemic range, when CL is actively modulating insulin delivery.
Conclusions: Consistent accuracy of the CGM within the euglycemic–hyperglycemic range using the Freestyle Navigator II was observed and supports its use in home CL studies. Our results may contribute toward establishing normative CGM performance criteria for unsupervised home use of CL.
doi:10.1089/dia.2015.0062
PMCID: PMC4649721  PMID: 26241693
3.  Lactobacillus casei Shirota Supplementation Does Not Restore Gut Microbiota Composition and Gut Barrier in Metabolic Syndrome: A Randomized Pilot Study 
PLoS ONE  2015;10(10):e0141399.
Metabolic syndrome is associated with disturbances in gut microbiota composition. We aimed to investigate the effect of Lactobacillus casei Shirota (LcS) on gut microbiota composition, gut barrier integrity, intestinal inflammation and serum bile acid profile in metabolic syndrome. In a single-centre, prospective, randomised controlled pilot study, 28 subjects with metabolic syndrome received either LcS for 12 weeks (n = 13) or no LcS (n = 15). Data were compared to healthy controls (n = 16). Gut microbiota composition was characterised from stool using 454 pyrosequencing of 16S rRNA genes. Serum bile acids were quantified by tandem mass spectrometry. Zonulin and calprotectin were measured in serum and stool by ELISA. Bacteroidetes/Firmicutes ratio was significantly higher in healthy controls compared to metabolic syndrome but was not influenced by LcS. LcS supplementation led to enrichment of Parabacteroides. Zonulin and calprotectin were increased in metabolic syndrome stool samples but not influenced by LcS supplementation. Serum bile acids were similar to controls and not influenced by LcS supplementation. Metabolic syndrome is associated with a higher Bacteroidetes/Firmicutes ratio and gut barrier dysfunction but LcS was not able to change this. LcS administration was associated with subtle microbiota changes at genus level.
Trial Registration
ClinicalTrials.gov NCT01182844
doi:10.1371/journal.pone.0141399
PMCID: PMC4625062  PMID: 26509793
4.  Standardized Glycemic Management with a Computerized Workflow and Decision Support System for Hospitalized Patients with Type 2 Diabetes on Different Wards 
Diabetes Technology & Therapeutics  2015;17(10):685-692.
Abstract
Background: This study investigated the efficacy, safety, and usability of standardized glycemic management by a computerized decision support system for non-critically ill hospitalized patients with type 2 diabetes on four different wards.
Materials and Methods: In this open, noncontrolled intervention study, glycemic management of 99 patients with type 2 diabetes (62% acute admissions; 41 females; age, 67±11 years; hemoglobin A1c, 65±21 mmol/mol; body mass index, 30.4±6.5 kg/m2) on clinical wards (Cardiology, Endocrinology, Nephrology, Plastic Surgery) of a tertiary-care hospital was guided by GlucoTab® (Joanneum Research GmbH [Graz, Austria] and Medical University of Graz [Graz, Austria]), a mobile decision support system providing automated workflow support and suggestions for insulin dosing to nurses and physicians.
Results: Adherence to insulin dosing suggestions was high (96.5% bolus, 96.7% basal). The primary outcome measure, percentage of blood glucose (BG) measurements in the range of 70–140 mg/dL, occurred in 50.2±22.2% of all measurements. The overall mean BG level was 154±35 mg/dL. BG measurements in the ranges of 60–70 mg/dL, 40–60 mg/dL, and <40 mg/dL occurred in 1.4%, 0.5%, and 0.0% of all measurements, respectively. A regression analysis showed that acute admission to the Cardiology Ward (+30 mg/dL) and preexisting home insulin therapy (+26 mg/dL) had the strongest impact on mean BG. Acute admission to other wards had minor effects (+4 mg/dL). Ninety-one percent of the healthcare professionals felt confident with GlucoTab, and 89% believed in its practicality and 80% in its ability to prevent medication errors.
Conclusions: An efficacious, safe, and user-accepted implementation of GlucoTab was demonstrated. However, for optimized personalized patient care, further algorithm modifications are required.
doi:10.1089/dia.2015.0027
PMCID: PMC4575539  PMID: 26355756
5.  Opposing roles of Prostaglandin D2 receptors in ulcerative colitis 
Pro-resolution functions were reported for Prostaglandin D2 (PGD2) in colitis, but the role of its two receptors, DP and in particular CRTH2 are less well defined. We investigated DP and CRTH2 expression and function during human and murine ulcerative colitis (UC). Expression of receptors was measured by flow cytometry on peripheral blood leukocytes, and by immunohistochemistry and immunoblotting in colon biopsies of patients with active UC and healthy individuals. Receptor involvement in UC was evaluated in a mouse model of DSS colitis. DP and CRTH2 expression changed in leukocytes of patients with active UC in a differential manner. In UC patients, DP showed higher expression in neutrophils but lower in monocytes as compared to control subjects. In contrast, CRTH2 was decreased in eosinophils, NK and CD3+ T cells but not in monocytes and CD3+/CD4+ T cells. The decrease of CRTH2 on blood eosinophils clearly correlated with disease activity. DP correlated positively with disease activity in eosinophils but inversely in neutrophils. CRTH2 internalized upon treatment with PGD2 and 11-dehydroTXB2 in eosinophils of controls. Biopsies of UC patients revealed an increase of CRTH2-positive cells in the colonic mucosa and high CRTH2 protein content. The CRTH2 antagonist CAY10595 improved while the DP antagonist MK0524 worsened inflammation in murine colitis. DP and CRTH2 play differential roles in UC. Although expression of CRTH2 on blood leukocytes is downregulated in UC, CRTH2 is present in colon tissue where it may contribute to inflammation whereas DP likely promotes anti-inflammatory actions.
doi:10.4049/jimmunol.1303484
PMCID: PMC4121674  PMID: 24929001
inflammatory bowel disease; eicosanoids; prostanoids; pro-inflammatory; DSS colitis
6.  Enhanced Absorption of Insulin Aspart as the Result of a Dispersed Injection Strategy Tested in a Randomized Trial in Type 1 Diabetic Patients 
Diabetes Care  2013;36(4):780-785.
OBJECTIVE
We investigated the impact of two different injection strategies on the pharmacokinetics and pharmacodynamics of insulin aspart in vivo in an open-label, two-period crossover study and verified changes in the surface-to-volume ratio ex vivo.
RESEARCH DESIGN AND METHODS
Before the clinical trial, insulin aspart was injected ex vivo into explanted human abdominal skin flaps. The surface-to-volume ratio of the subcutaneous insulin depot was assessed by microfocus computed tomography that compared 1 bolus of 18 IU with 9 dispersed boluses of 2 IU. These two injection strategies were then tested in vivo, in 12 C-peptide–negative type 1 diabetic patients in a euglycemic glucose clamp (glucose target 5.5 ± 1.1 mmol/L) for 8 h after the first insulin administration.
RESULTS
The ex vivo experiment showed a 1.8-fold higher mean surface-to-volume ratio for the dispersed injection strategy. The maximum glucose infusion rates (GIR) were similar for the two strategies (10 ± 4 vs. 9 ± 4; P = 0.5); however, times to reach maximum GIR and 50% and 10% of the maximum GIR were significantly reduced by using the 9 × 2 IU strategy (68 ± 33 vs. 127 ± 93 min; P = 0.01; 38 ± 9 vs. 49 ± 16 min; P < 0.01; 23 ± 6 vs. 30 ± 10 min; P < 0.05). For 9 × 2 IU, the area under the GIR curve was greater during the first 60 min (219 ± 89 vs. 137 ± 75; P < 0.01) and halved until maximum GIR (242 ± 183 vs. 501 ± 396; P < 0.01); however, it was similar across the whole study period (1,361 ± 469 vs. 1,565 ± 527; P = 0.08).
CONCLUSIONS
A dispersed insulin injection strategy enhanced the effect of a fast-acting insulin analog. The increased surface-to-volume ratio of the subcutaneous insulin depot can facilitate insulin absorption into the vascular system.
doi:10.2337/dc12-1319
PMCID: PMC3609526  PMID: 23193211
7.  Acetyl-coenzyme A 
Autophagy  2014;10(7):1335-1337.
As the major lysosomal degradation pathway, autophagy represents the guardian of cellular homeostasis, removing damaged and potentially harmful material and replenishing energy reserves in conditions of starvation. Given its vast physiological importance, autophagy is crucially involved in the process of aging and associated pathologies. Although the regulation of autophagy strongly depends on nutrient availability, specific metabolites that modulate autophagic responses are poorly described. Recently, we revealed nucleo-cytosolic acetyl-coenzyme A (AcCoA) as a phylogenetically conserved inhibitor of starvation-induced and age-associated autophagy. AcCoA is the sole acetyl-group donor for protein acetylation, explaining why pharmacological or genetic manipulations that modify the concentrations of nucleo-cytosolic AcCoA directly affect the levels of protein acetylation. The acetylation of histones and cytosolic proteins inversely correlates with the rate of autophagy in yeast and mammalian cells, respectively, despite the fact that the routes of de novo AcCoA synthesis differ across phyla. Thus, we propose nucleo-cytosolic AcCoA to act as a conserved metabolic rheostat, linking the cellular metabolic state to the regulation of autophagy via effects on protein acetylation.
doi:10.4161/auto.28919
PMCID: PMC4203557  PMID: 24904996
autophagy; aging; acetyl-coenzyme A; histone acetylation; transcription; epigenetics; ATG
8.  A histone point mutation that switches on autophagy 
Autophagy  2014;10(6):1143-1145.
The multifaceted process of aging inevitably leads to disturbances in cellular metabolism and protein homeostasis. To meet this challenge, cells make use of autophagy, which is probably one of the most important pathways preserving cellular protection under stressful conditions. Thus, efficient autophagic flux is required for healthy aging in many if not all eukaryotic organisms. The regulation of autophagy itself is affected by changing metabolic conditions, but the precise metabolic circuitries are poorly understood. Recently, we found that the nucleocytosolic pool of acetyl-coenzyme A (AcCoA) functions as a major and dominant suppressor of cytoprotective autophagy during aging. Here, we propose an epigenetic mechanism for AcCoA-mediated autophagy suppression that causally involves the regulation of histone acetylation and changes in the autophagy-relevant transcriptome.
doi:10.4161/auto.28767
PMCID: PMC4091175  PMID: 24879160
acetyl-coenzyme A; aging; ATG; autophagy; epigenetic; histone acetylation; transcription
9.  Assessing the effectiveness of 3 months day and night home closed-loop insulin delivery in adults with suboptimally controlled type 1 diabetes: a randomised crossover study protocol 
BMJ Open  2014;4(9):e006075.
Introduction
Despite therapeutic advances, many people with type 1 diabetes are still unable to achieve optimal glycaemic control, limited by the occurrence of hypoglycaemia. The objective of the present study is to determine the effectiveness of day and night home closed-loop over the medium term compared with sensor-augmented pump therapy in adults with type 1 diabetes and suboptimal glycaemic control.
Methods and analysis
The study will adopt an open label, three-centre, multinational, randomised, two-period crossover study design comparing automated closed-loop glucose control with sensor augmented insulin pump therapy. The study will aim for 30 completed participants. Eligible participants will be adults (≥18 years) with type 1 diabetes treated with insulin pump therapy and suboptimal glycaemic control (glycated haemoglobin (HbA1c) ≥7.5% (58 mmol/mmol) and ≤10% (86 mmol/mmol)). Following a 4-week optimisation period, participants will undergo a 3-month use of automated closed-loop insulin delivery and sensor-augmented pump therapy, with a 4–6 week washout period in between. The order of the interventions will be random. All analysis will be conducted on an intention to treat basis. The primary outcome is the time spent in the target glucose range from 3.9 to 10.0 mmol/L based on continuous glucose monitoring levels during the 3 months free living phase. Secondary outcomes include HbA1c changes; mean glucose and time spent above and below target glucose levels. Further, participants will be invited at baseline, midpoint and study end to participate in semistructured interviews and complete questionnaires to explore usability and acceptance of the technology, impact on quality of life and fear of hypoglycaemia.
Ethics and dissemination
Ethical approval has been obtained at all sites. Before screening, all participants will be provided with oral and written information about the trial. The study will be disseminated by peer-review publications and conference presentations.
Trial registration number
NCT01961622 (ClinicalTrials.gov).
doi:10.1136/bmjopen-2014-006075
PMCID: PMC4158197  PMID: 25186158
Closed-loop; Type 1 diabetes; Continuous glucose monitoring; Artificial pancreas; Model predictive control
10.  Glucose control in intensive care: usability, efficacy and safety of Space GlucoseControl in two medical European intensive care units 
Background
The Space GlucoseControl system (SGC) is a nurse-driven, computer-assisted device for glycemic control combining infusion pumps with the enhanced Model Predictive Control algorithm (B. Braun, Melsungen, Germany). We aimed to investigate the performance of the SGC in medical critically ill patients.
Methods
Two open clinical investigations in tertiary centers in Graz, Austria and Zurich, Switzerland were performed. Efficacy was assessed by percentage of time within the target range (4.4-8.3 mmol/L; primary end point), mean blood glucose, and sampling interval. Safety was assessed by the number of hypoglycemic episodes (≤2.2 mmol/L) and the percentage of time spent below this cutoff level. Usability was analyzed with a standardized questionnaire given to involved nursing staff after the trial.
Results
Forty medical critically ill patients (age, 62 ± 15 years; body mass index, 30.0 ± 8.9 kg/m2; APACHE II score, 24.8 ± 5.4; 27 males; 8 with diabetes) were included for a period of 6.5 ± 3.7 days (n = 20 in each center). The primary endpoint (time in target range 4.4 to 8.3 mmol/l) was reached in 88.3% ± 9.3 of the time and mean arterial blood glucose was 6.7 ± 0.4 mmol/l. The sampling interval was 2.2 ± 0.4 hours. The mean daily insulin dose was 87.2 ± 64.6 IU. The adherence to the given insulin dose advice was high (98.2%). While the percentage of time spent in a moderately hypoglycemic range (2.2 to 3.3 mmol/L) was low (0.07 ± 0.26% of the time), one severe hypoglycemic episode (<2.2 mmol/L) occurred (2.5% of patients or 0.03% of glucose readings).
Conclusions
SGC is a safe and efficient method to control blood glucose in critically ill patients as assessed in two European medical intensive care units.
doi:10.1186/1472-6823-14-62
PMCID: PMC4118658  PMID: 25074071
Tight glycemic control; Critical illness; Critically ill patients; Protocol; Computer-assisted glycemic control; Insulin infusion protocol; Glucose control in intensive care
11.  The effect of terbutaline on the absorption of pulmonary administered insulin in subjects with asthma 
AIM
To investigate the effect of prior administration of a bronchodilator on the absorption of inhaled insulin in people with asthma treated with inhaled corticosteroids.
METHODS
A single-centre, randomized, open-label, two-period cross-over trial was carried out in 41 nondiabetic subjects with asthma treated with inhaled steroids, with reversible bronchoconstriction (Rev+; n= 25) or without reversible bronchoconstriction (Rev−; n= 16). A dose of 0.10 U kg−1 inhaled human insulin was administered on each dosing day with or without prior administration of the bronchodilator terbutaline (in random order).
RESULTS
Prior administration of terbutaline led to a 44% increase in absorption of insulin over 6 h for the Rev+ group compared with no prior administration of bronchodilator [ratio (95% confidence interval) 1.44 (1.13, 1.82), P= 0.004], whereas no effect was seen for the Rev− or the whole group. The maximum insulin concentration (Cmax) increased by 34% for the Rev+ group (P = 0.018) and 17% for the whole group (P= 0.046), whereas no significant effect of prior terbutaline administration was seen for Rev−. The time to Cmax was not significantly different for the Rev+ group, whereas it was approximately 30% longer after bronchodilator administration for the Rev− group (P= 0.044) and the whole group (P= 0.032).
CONCLUSIONS
In people with asthma and reversible bronchoconstriction, the administration of a bronchodilator prior to administration of inhaled insulin led to increased absorption of insulin, whereas no effect on insulin absorption in subjects without significant reversibility could be detected.
doi:10.1111/j.1365-2125.2009.03573.x
PMCID: PMC2829697  PMID: 20233198
aerosol; asthma; drug safety; insulin absorption; pharmacodynamics; pharmacokinetics
13.  Multiple risk factor intervention reduces carotid atherosclerosis in patients with type 2 diabetes 
Background
Patients with rapid progression of carotid intima media thickness (CIMT) were shown to have a higher future risk for cardiovascular events.
The aim of this study was to investigate the impact of multiple risk factor intervention on CIMT progression and to establish whether new cardiovascular surrogate measurements would allow prediction of CIMT changes.
Materials and methods
In this prospective, open, 2-years study, we included 97 patients with type 2 diabetes and at least two insufficiently treated cardiovascular risk factors, i.e. HbA1c > 7.5% (58 mmol/mol); LDL-cholesterol >3.1 mmol/l or blood pressure >140/90 mmHg. Treatment was intensified according to current guidelines over 3 months with the aim to maintain intensification over 2 years.
The primary outcome was the change in CIMT after 2 years. We also assessed markers of mechanical and biochemical endothelial function and endothelial progenitor cells before and after 3 months of treatment intensification. For testing differences between before and after multifactorial treatment measurements we used either the paired student’s t-test or the Wilcoxon signed-rank test, depending on the distribution of the data. Additional, explorative statistical data analysis was done on CIMT progression building a linear multivariate regression model.
Results
Blood glucose, lipids and blood pressure significantly improved during the first 3 months of intensified treatment, which was sustained over the 2-year study duration. Mean CIMT significantly decreased from baseline to 2 year (0.883 ± 0.120 mm vs. 0.860 ± 0.130 mm; p = 0.021). None of the investigated surrogate measures, however, was able to predict changes in IMT early after treatment intensification.
Conclusions
Intensification of risk factor intervention in type 2 diabetes results in CIMT regression over a period of 2 years. None of the biomarkers used including endothelial function parameters or endothelial progenitor cells turned out to be useful to predict CIMT changes.
Trial registration
Clinical Trial Registration – Unique identifier: NCT00660790
doi:10.1186/1475-2840-13-95
PMCID: PMC4041351  PMID: 24884694
Intensified risk factor intervention; Carotid intima media thickness; Type 2 diabetes; Cardiovascular surrogate measurements; Carotid atherosclerosis
14.  Assessment of Blood-Brain Barrier Function and the Neuroinflammatory Response in the Rat Brain by Using Cerebral Open Flow Microperfusion (cOFM) 
PLoS ONE  2014;9(5):e98143.
Blood-brain barrier (BBB) impairment in systemic inflammation leads to neuroinflammation. Several factors including cytokines, chemokines and signal transduction molecules are implicated in BBB dysfunction in response to systemic inflammation. Here, we have adopted a novel in vivo technique; namely, cerebral open flow microperfusion (cOFM), to perform time-dependent cytokine analysis (TNF-alpha, IL-6 and IL-10) in the frontal cortex of the rat brain in response to a single peripheral administration of lipopolysaccharide (LPS). In parallel, we monitored BBB function using sodium fluorescein as low molecular weight reporter in the cOFM sample. In response to the systemic LPS administration, we observed a rapid increase of TNF-alpha in the serum and brain, which coincides with the BBB disruption. Brain IL-6 and IL-10 synthesis was delayed by approximately 1 h. Our data demonstrate that cOFM can be used to monitor changes in brain cytokine levels and BBB disruption in a rat sepsis model.
doi:10.1371/journal.pone.0098143
PMCID: PMC4031165  PMID: 24852285
15.  Vitamin D status and its association with season, hospital and sepsis mortality in critical illness 
Critical Care  2014;18(2):R47.
Introduction
Vitamin D plays a key role in immune function. Deficiency may aggravate the incidence and outcome of infectious complications in critically ill patients. We aimed to evaluate the prevalence of vitamin D deficiency and the correlation between serum 25-hydroxyvitamin D (25(OH) D) and hospital mortality, sepsis mortality and blood culture positivity.
Methods
In a single-center retrospective observational study at a tertiary care center in Graz, Austria, 655 surgical and nonsurgical critically ill patients with available 25(OH) D levels hospitalized between September 2008 and May 2010 were included. Cox regression analysis adjusted for age, gender, severity of illness, renal function and inflammatory status was performed. Vitamin D levels were categorized by month-specific tertiles (high, intermediate, low) to reflect seasonal variation of serum 25(OH) D levels.
Results
Overall, the majority of patients were vitamin D deficient (<20 ng/ml; 60.2%) or insufficient (≥20 and <30 ng/dl; 26.3%), with normal 25(OH) D levels (>30 ng/ml) present in only 13.6%. The prevalence of vitamin D deficiency and mean 25(OH) D levels was significantly different in winter compared to summer months (P <0.001). Hospital mortality was 20.6% (135 of 655 patients). Adjusted hospital mortality was significantly higher in patients in the low (hazard ratio (HR) 2.05, 95% confidence interval (CI) 1.31 to 3.22) and intermediate (HR 1.92, 95% CI 1.21 to 3.06) compared to the high tertile. Sepsis was identified as cause of death in 20 of 135 deceased patients (14.8%). There was no significant association between 25(OH) D and C-reactive protein (CRP), leukocyte count or procalcitonin levels. In a subgroup analysis (n = 244), blood culture positivity rates did not differ between tertiles (23.1% versus 28.2% versus 17.1%, P = 0.361).
Conclusions
Low 25(OH) D status is significantly associated with mortality in the critically ill. Intervention studies are needed to investigate the effect of vitamin D substitution on mortality and sepsis rates in this population.
doi:10.1186/cc13790
PMCID: PMC4057427  PMID: 24661739
16.  Long-Term Implanted cOFM Probe Causes Minimal Tissue Reaction in the Brain 
PLoS ONE  2014;9(3):e90221.
This study investigated the histological tissue reaction to long-term implanted cerebral open flow microperfusion (cOFM) probes in the frontal lobe of the rat brain. Most probe-based cerebral fluid sampling techniques are limited in application time due to the formation of a glial scar that hinders substance exchange between brain tissue and the probe. A glial scar not only functions as a diffusion barrier but also alters metabolism and signaling in extracellular brain fluid. cOFM is a recently developed probe-based technique to continuously sample extracellular brain fluid with an intact blood-brain barrier. After probe implantation, a 2 week healing period is needed for blood-brain barrier reestablishment. Therefore, cOFM probes need to stay in place and functional for at least 15 days after implantation to ensure functionality. Probe design and probe materials are optimized to evoke minimal tissue reaction even after a long implantation period. Qualitative and quantitative histological tissue analysis revealed no continuous glial scar formation around the cOFM probe 30 days after implantation and only a minor tissue reaction regardless of perfusion of the probe.
doi:10.1371/journal.pone.0090221
PMCID: PMC3951198  PMID: 24621608
17.  Nucleocytosolic Depletion of the Energy Metabolite Acetyl-Coenzyme A Stimulates Autophagy and Prolongs Lifespan 
Cell Metabolism  2014;19(3):431-444.
Summary
Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation.
Graphical Abstract
Highlights
•Acetyl-CoA (AcCoA) metabolism regulates autophagy during aging•Autophagy regulation by AcCoA metabolism acts downstream of nutrient signaling•Brain-specific knockdown of Drosophila AcCoA synthetase prolongs lifespan•Histone point mutations permanently activate autophagy during aging
Autophagy plays a crucial role in healthy aging. By blocking mitochondrial AcCoA production, Eisenberg et al. show that accumulation of nucleocytosolic AcCoA inhibits autophagy and reduces lifespan through a conserved epigenetic mechanism involving histone acetylation of specific autophagy genes in yeast and flies.
doi:10.1016/j.cmet.2014.02.010
PMCID: PMC3988959  PMID: 24606900
18.  Periodic Extraction of Interstitial Fluid from the Site of Subcutaneous Insulin Infusion for the Measurement of Glucose: A Novel Single-Port Technique for the Treatment of Type 1 Diabetes Patients 
Abstract
Background
Treatment of type 1 diabetes patients could be simplified if the site of subcutaneous insulin infusion could also be used for the measurement of glucose. This study aimed to assess the agreement between blood glucose concentrations and glucose levels in the interstitial fluid (ISF) that is extracted from the insulin infusion site during periodic short-term interruptions of continuous subcutaneous insulin infusion (CSII).
Subjects and Methods
A perforated cannula (24 gauge) was inserted into subcutaneous adipose tissue of C-peptide-negative type 1 diabetes subjects (n=13) and used alternately to infuse rapid-acting insulin (100 U/mL) and to extract ISF glucose during a fasting period and after ingestion of a standard oral glucose load (75 g).
Results
Although periodically interrupted for extracting glucose (every hour for approximately 10 min), insulin infusion with the cannula was adequate to achieve euglycemia during fasting and to restore euglycemia after glucose ingestion. Furthermore, the ISF-derived estimates of plasma glucose levels agreed well with plasma glucose concentrations. Correlation coefficient and median absolute relative difference values were found to be 0.95 and 8.0%, respectively. Error grid analysis showed 99.0% of all ISF glucose values within clinically acceptable Zones A and B (83.5% Zone A, 15.5% Zone B).
Conclusions
Results show that ISF glucose concentrations measured at the insulin infusion site during periodic short-term interruptions of CSII closely reflect blood glucose levels, thus suggesting that glucose monitoring and insulin delivery may be performed alternately at the same tissue site. A single-port device of this type could be used to simplify and improve glucose management in diabetes.
doi:10.1089/dia.2012.0173
PMCID: PMC3540899  PMID: 23126579
19.  The effect of exercise on the absorption of inhaled human insulin in healthy volunteers 
Aims
To investigate the effect of moderate exercise on the absorption of inhaled insulin.
Methods
A single-centre, randomized, open-label, three-period cross-over trial was carried out in 12 nonsmoking healthy subjects. A dose of 3.5 mg inhaled human insulin was administered via a nebulizer and followed in random order by either 1) no exercise (NOEX), 2) 30 min exercise starting immediately after dosing (EX0), or 3) 30 min exercise starting 30 min after dosing (EX30). The study was carried out as a 10 h euglycaemic glucose clamp (90 mg dl−1 (5.0 mmol l−1)).
Results
The absorption of insulin over the first 2 h after start of exercise was 16% increased for EX0 (ratio (95%CI) 1.16 (1.04, 1.30), P = 0.01) and 20% increased for EX30 (1.20 (1.05, 1.36), P < 0.01), both compared with NOEX; the overall insulin absorption during 6 h and 10 h after dosing was not influenced by exercise. The maximum insulin concentration (Cmax) increased by 32% for EX0 and 35% for EX30 (both P < 0.01) compared with NOEX, while the time to Cmax was 31 min faster for EX0 (P < 0.01), but not significantly different after EX30, compared with NOEX.
Conclusions
A significant and clinically relevant increase of insulin absorption over the first 2 h after the beginning of exercise was observed. Until data from studies using the specific insulin inhalers exists, patients using inhaled insulin should be made aware of a potential increased absorption and higher concentration of insulin in connection with exercise.
What is already known about this subject Exercise is known to affect absorption of other inhaled substances, but so far there are no reports on the effect of exercise on the absorption of inhaled insulin in humans.
What this paper adds This report is the first to investigate the effect of exercise on the absorption of inhaled insulin.In this study in healthy volunteers we found that exercise early after dosing increased absorption (15–20%) of inhaled insulin over the first 2 h after start of exercise, with an approximately 30% increase in maximal insulin concentration, and unchanged overall absorption.
doi:10.1111/j.1365-2125.2007.03000.x
PMCID: PMC2291224  PMID: 17764475
administration; aerosol; drug safety; inhalation; lung; pharmacodynamics; pharmacokinetics
20.  Distribution of CD4pos -, CD8pos – and Regulatory T Cells in the Upper and Lower Gastrointestinal Tract in Healthy Young Subjects 
PLoS ONE  2013;8(11):e80362.
The gastrointestinal immune system is involved in the development of several autoimmune-mediated diseases, including inflammatory bowel disease, multiple sclerosis, and type 1 diabetes mellitus. Alterations in T-cell populations, especially regulatory T cells (Tregs), are often evident in patients suffering from these diseases. To be able to detect changes in T-cell populations in diseased tissue, it is crucial to investigate T-cell populations in healthy individuals, and to characterize their variation among different regions of the gastrointestinal (GI) tract. While limited data exist, quantitative data on biopsies systematically drawn from various regions of the GI tract are lacking, particularly in healthy young humans. In this report, we present the first systematic assessment of how T cells—including Tregs—are distributed in the gastrointestinal mucosa throughout the GI tract of healthy young humans by means of multi-parameter FACS analysis. Gastroduodenoscopy and colonoscopy were performed on 16 healthy volunteers aged between 18 and 32. Biopsies were drawn from seven GI regions, and were used to determine the frequencies of CD8+-, CD4+- and Tregs in the gastrointestinal mucosa by means of multi-parameter FACS analysis. Our data show that there is significant variation in the baseline T-cell landscape along the healthy human gastrointestinal tract, and that mucosal T-cell analyses from a single region should not be taken as representative of the entire gastrointestinal tract. We show that certain T-cell subsets in the gastrointestinal mucosa vary significantly among regions; most notably, that Tregs are enriched in the appendiceal orifice region and the ascending colon, and that CD8pos T cells are enriched in the gastric mucosa.
doi:10.1371/journal.pone.0080362
PMCID: PMC3827200  PMID: 24265815
21.  Insulin degludec is not associated with a delayed or diminished response to hypoglycaemia compared with insulin glargine in type 1 diabetes: a double-blind randomised crossover study 
Diabetologia  2013;57(1):40-49.
Aims/hypothesis
Insulin degludec (Des(B30)LysB29(γ-Glu Nε-hexadecandioyl) human insulin; IDeg) is a new basal insulin with an ultra-long flat action profile. The acute physiological responses to hypoglycaemia with IDeg and insulin glargine (A21Gly,B31Arg,B32Arg human insulin; IGlar) were compared.
Methods
Twenty-eight adult type 1 diabetic patients with normal hypoglycaemia awareness (age = 41 ± 12 years, HbA1c = 7.8 ± 0.6% [62.8 ± 7 mmol/mol]) were randomised to once-daily IDeg or IGlar for 5 days in a two-period crossover design. Participants and research staff were blinded to group assignment. Patients were assigned the lowest available randomisation number from a set of blinded randomisation codes provided by the trial sponsor. Hypoglycaemia was induced by administering three times the usual daily insulin dose at midnight on day 5. Plasma glucose (PG) was stabilised by glucose clamp (5.5 mmol/l) for 7–9 h post dosing. Next morning, PG was allowed to decrease stepwise from 5.5 to 3.5 mmol/l (maintained for 30 min) to 2.5 mmol/l (for 15 min). PG was then increased to 3.9 mmol/l (for 120 min), before being returned to baseline. Hypoglycaemic symptom score (HSS), hypoglycaemic awareness, cognitive function, counter-regulatory hormones and vital signs were assessed during each glucose plateau. The primary analysis was to compare IDeg vs IGlar with respect to HSS at nadir PG concentration (2.5 mmol/l).
Results
The full analysis set for treatment comparisons comprised data from all 28 exposed patients. Rates of PG decline and PG at nadir were similar for IDeg and IGlar. No treatment differences in HSS (estimated difference: 0.17 [95% CI −1.71, 2.05]; p > 0.05), cognitive function or awareness were observed at any time. Growth hormone and cortisol responses during hypoglycaemia were greater with IDeg than IGlar (AUC treatment ratio [IDeg/IGlar]: 2.44 [1.30, 4.60], p < 0.01; and 1.23 [1.01, 1.50]; p < 0.05), and adrenaline (epinephrine) responses trended higher (1.40 [0.96, 2.04], p = 0.07). The rates of recovery from hypoglycaemia were similar.
Conclusions/interpretation
IDeg and IGlar elicit comparable symptomatic and cognitive responses to induced hypoglycaemia. IDeg may elicit a moderately greater endocrine response, but times to PG recovery were similar for the two insulins.
Trial registration
ClinicalTrials.gov NCT01002768.
Funding
Novo Nordisk.
doi:10.1007/s00125-013-3056-0
PMCID: PMC3855490  PMID: 24057153
Counter-regulation; Degludec; Glargine; Hormones; Hypoglycaemia; Type 1 diabetes
22.  Vitamin D and Immune Function 
Nutrients  2013;5(7):2502-2521.
Vitamin D metabolizing enzymes and vitamin D receptors are present in many cell types including various immune cells such as antigen-presenting-cells, T cells, B cells and monocytes. In vitro data show that, in addition to modulating innate immune cells, vitamin D also promotes a more tolerogenic immunological status. In vivo data from animals and from human vitamin D supplementation studies have shown beneficial effects of vitamin D on immune function, in particular in the context of autoimmunity. In this review, currently available data are summarized to give an overview of the effects of vitamin D on the immune system in general and on the regulation of inflammatory responses, as well as regulatory mechanisms connected to autoimmune diseases particularly in type 1 diabetes mellitus.
doi:10.3390/nu5072502
PMCID: PMC3738984  PMID: 23857223
vitamin D; autoimmunity; immune cells; adaptive immunity; innate immunity; cholecalciferol; calcitriol; 25(OH)D
23.  Mutations at Ser331 in the HSN type I gene SPTLC1 are associated with a distinct syndromic phenotype 
Mutations in the serine palmitoyltransferase subunit 1 (SPTLC1) gene are the most common cause of hereditary sensory neuropathy type 1 (HSN1). Here we report the clinical and molecular consequences of a particular mutation (p.S331Y) in SPTLC1 affecting a patient with severe, diffuse muscle wasting and hypotonia, prominent distal sensory disturbances, joint hypermobility, bilateral cataracts and considerable growth retardation. Normal plasma sphingolipids were unchanged but 1-deoxy-sphingolipids were significantly elevated. In contrast to other HSN patients reported so far, our findings strongly indicate that mutations at amino acid position Ser331 of the SPTLC1 gene lead to a distinct syndrome.
Highlights
•Novel mutation associated with a distinct new phenotype.•Most interesting because of possible upcoming therapeutic options.•Elevated 1-deoxy-sphingolipids levels.
doi:10.1016/j.ejmg.2013.02.002
PMCID: PMC3682180  PMID: 23454272
HSN; HSAN; SPTLC1; Cataract; Hereditary neuropathy
24.  Comparison of two in vitro systems to assess cellular effects of nanoparticles-containing aerosols 
Inhalation treatment with nanoparticle containing aerosols appears a promising new therapeutic option but new formulations have to be assessed for efficacy and toxicity. We evaluated the utility of a VITRO-CELL®6 PT-CF + PARI LC SPRINT® Baby Nebulizer (PARI BOY) system compared with a conventional MicroSprayer. A549 cells were cultured in the air–liquid interface, exposed to nanoparticle aerosols and characterized by measurement of transepithelial electrical resistance and staining for tight junction proteins. Deposition and distribution rates of polystyrene particles and of carbon nanotubes on the cells were assessed. In addition, cytotoxicity of aerosols containing polystyrene particles was compared with cytotoxicity of polystyrene particles in suspension tested in submersed cultures. Exposure by itself in both exposure systems did not damage the cells. Deposition rates of aerosolized polystyrene particles were about 700 times and that of carbon nanotubes about 4 times higher in the MicroSprayer than in the VITROCELL®6 PT-CF system. Cytotoxicity of amine-functionalized polystyrene nanoparticles was significantly higher when applied as an aerosol on cell cultured in air–liquid interface culture compared with nanoparticle suspensions tested in submersed culture. The higher cytotoxicity of aerosolized nanoparticles underscores the importance of relevant exposure systems.
doi:10.1016/j.tiv.2012.08.008
PMCID: PMC3514486  PMID: 22906573
Nanoparticles; Exposure systems; Inhalation treatment; Nanotoxicology
25.  Comparison of two in vitro systems to assess cellular effects of nanoparticles-containing aerosols 
Toxicology in Vitro  2013;27-360(1):409-417.
Highlights
► A new VITROCELL – Pariboy system was evaluated for testing of aerosolized NPs. ► Deposition rates differed between marker compounds and NPs. ► The manual aerosolizer MicroSprayer was suitable for cytotoxicity testing of NPs. ► Polystyrene nanoparticles acted more cytotoxic as aerosols than as suspensions.
Inhalation treatment with nanoparticle containing aerosols appears a promising new therapeutic option but new formulations have to be assessed for efficacy and toxicity. We evaluated the utility of a VITROCELL®6 PT-CF + PARI LC SPRINT® Baby Nebulizer (PARI BOY) system compared with a conventional MicroSprayer. A549 cells were cultured in the air–liquid interface, exposed to nanoparticle aerosols and characterized by measurement of transepithelial electrical resistance and staining for tight junction proteins. Deposition and distribution rates of polystyrene particles and of carbon nanotubes on the cells were assessed. In addition, cytotoxicity of aerosols containing polystyrene particles was compared with cytotoxicity of polystyrene particles in suspension tested in submersed cultures. Exposure by itself in both exposure systems did not damage the cells. Deposition rates of aerosolized polystyrene particles were about 700 times and that of carbon nanotubes about 4 times higher in the MicroSprayer than in the VITROCELL®6 PT-CF system. Cytotoxicity of amine-functionalized polystyrene nanoparticles was significantly higher when applied as an aerosol on cell cultured in air–liquid interface culture compared with nanoparticle suspensions tested in submersed culture. The higher cytotoxicity of aerosolized nanoparticles underscores the importance of relevant exposure systems.
doi:10.1016/j.tiv.2012.08.008
PMCID: PMC3514486  PMID: 22906573
ALI, air liquid interface; FS, FluoSpheres; FBS, fetal bovine serum; PBS, phosphate buffered saline; DMEM, Dulbecco’s minimal essential medium; ZO-1, zona occludens protein 1; TEER, transepithelial electrical resistance; Nanoparticles; Exposure systems; Inhalation treatment; Nanotoxicology

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