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.
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.
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.
Clinical Trial Registration – Unique identifier:
Intensified risk factor intervention; Carotid intima media thickness; Type 2 diabetes; Cardiovascular surrogate measurements; Carotid atherosclerosis
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.
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).
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.
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.
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).
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).
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.
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.
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.
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).
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.
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.
Counter-regulation; Degludec; Glargine; Hormones; Hypoglycaemia; Type 1 diabetes
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.
vitamin D; autoimmunity; immune cells; adaptive immunity; innate immunity; cholecalciferol; calcitriol; 25(OH)D
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.
•Novel mutation associated with a distinct new phenotype.•Most interesting because of possible upcoming therapeutic options.•Elevated 1-deoxy-sphingolipids levels.
HSN; HSAN; SPTLC1; Cataract; Hereditary neuropathy
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.
Nanoparticles; Exposure systems; Inhalation treatment; Nanotoxicology
► 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.
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
Spermidine is a naturally occurring polyamine involved in multiple biological processes, including DNA metabolism, autophagy and aging. Like other polyamines, spermidine is also indispensable for successful reproduction at several stages. However, a direct influence on the actual fertilization process, i.e., the fusion of an oocyte with a spermatocyte, remains uncertain. To explore this possibility, we established the mating process in the yeast Saccharomyces cerevisiae as a model for fertilization in higher eukaryotes. During human fertilization, the sperm capacitates and the acrosome reaction is necessary for penetration of the oocyte. Similarly, sexually active yeasts form a protrusion called “shmoo” as a prerequisite for mating. In this study, we demonstrate that pheromone-induced shmoo formation requires spermidine. In addition, we show that spermidine is essential for mating in yeast as well as for egg fertilization in the nematode Caenorhabditis elegans. In both cases, this occurs independently from autophagy. In synthesis, we identify spermidine as an important mating component in unicellular and multicellular model organisms, supporting an unprecedented evolutionary conservation of the mechanisms governing fertilization-related cellular fusion.
Caenorhabditis elegans; spermidine; mating; fertilization; Saccharomyces cerevisiae; shmoo; autophagy; sexual reproduction
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.
Vitamin D deficiency is associated with multiple adverse health outcomes including increased morbidity and mortality in the general population and in critically ill patients. However, no randomized controlled trial has evaluated so far whether treatment with sufficiently large doses of vitamin D can improve clinical outcome of patients in an intensive care setting.
The VITdAL@ICU trial is an investigator-initiated, non-commercial, double-blind, placebo-controlled randomized clinical trial. This study compares high-dose oral cholecalciferol (vitamin D3) versus placebo treatment in a mixed population of 480 critically ill patients with low 25-hydroxyvitamin-D levels at study enrollment (≤ 20ng/ml). Following an initial loading dose of 540,000 IU of vitamin D3, patients receive 90,000 IU of vitamin D3 on a monthly basis for 5 months. The study is designed to compare clinical outcome in the two study arms with the primary endpoint being length of hospital stay. Secondary endpoints include among others length of ICU stay, the percentage of patients with 25(OH)D levels > 30 ng/ml at day 7, ICU and hospital mortality and duration of mechanical ventilation. We describe here the VITdAL@ICU study protocol for the primary report.
This trial is designed to evaluate whether high-dose vitamin D3 is able to improve morbidity and mortality in a mixed population of adult critically ill patients and correct vitamin D deficiency safely.
Critical Illness; Vitamin D deficiency; Cholecalciferol; Vitamin D; Critical care; Intensive care; Vitamin D3
Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded.
CPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced.
Despite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs.
Nanoparticles; Lysosomes; Cathepsin B; Lysosomal sulfatase; Accumulation
To identify the disease-causing gene responsible for an autosomal dominantly inherited Charcot–Marie–Tooth neuropathy subtype in a family excluded for mutations in the common Charcot–Marie–Tooth genes, we used array-based sequence capture to simultaneously analyse the disease-linked protein coding exome at chromosome 14q32. A missense mutation in fibulin-5, encoding a widely expressed constituent of the extracellular matrix that has an essential role in elastic fibre assembly and has been shown to cause cutis laxa, was detected as the only novel non-synonymous sequence variant within the disease interval. Screening of 112 index probands with unclassified Charcot–Marie–Tooth neuropathies detected two further fibulin-5 missense mutations in two families with Charcot–Marie–Tooth disease and hyperextensible skin. Since fibulin-5 mutations have been described in patients with age-related macular degeneration, an additional 300 probands with exudative age-related macular degeneration were included in this study. Two further fibulin-5 missense mutations were identified in six patients. A mild to severe peripheral neuropathy was detected in the majority of patients with age-related macular degeneration carrying mutations in fibulin-5. This study identifies fibulin-5 as a gene involved in Charcot–Marie–Tooth neuropathies and reveals heterozygous fibulin-5 mutations in 2% of our patients with age-related macular degeneration. Furthermore, it adumbrates a new syndrome by linking concurrent pathologic alterations affecting peripheral nerves, eyes and skin to mutations in the fibulin-5 gene.
age-related macular degeneration; CMT; cutis laxa; fibulin-5; neuropathy
Distal hereditary motor neuropathy type V (dHMN-V) and Charcot–Marie–Tooth syndrome (CMT) type 2 presenting with predominant hand involvement, also known as CMT2D and Silver syndrome (SS) are rare phenotypically overlapping diseases which can be caused by mutations in the Berardinelli–Seip Congenital Lipodystrophy 2 (BSCL2) and in the glycyl-tRNA synthetase encoding (GARS) genes. Mutations in the heat-shock proteins HSPB1 and HSPB8 can cause related distal hereditary motor neuropathies (dHMN) and are considered candidates for dHMN-V, CMT2, and SS.
To define the frequency and distribution of mutations in the GARS, BSCL2, HSPB1 and HSPB8 genes we screened 33 unrelated sporadic and familial patients diagnosed as either dHMN-V, CMT2D or SS. Exon 3 of the BSCL2 gene was screened in further 69 individuals with an unclassified dHMN phenotype or diagnosed as hereditary spastic paraplegia (HSP) complicated by pure motor neuropathy.
Four patients diagnosed with dHMN-Vor SS carried known heterozygous BSCL2 mutations (N88S and S90L). In one dHMN-V patient we detected a putative GARS mutation (A57V). No mutations were detected in HSPB1 and HSPB8. The diagnostic yield gained in the series of 33 probands was 12% for BSCL2 mutations and 3% for GARS mutations. In the series of unclassified dHMN and complicated HSP cases no mutations were found.
Our data confirm that most likely only two mutations (N88S, S90L) in exon 3 of BSCL2 may lead to dHMN-V or SS phenotypes. Mutations in GARS, HSPB1 and HSPB8. are not a common cause of dHMN-V, SS and CMT2D. We would therefore suggest that a genetic testing of dHMN-V and SS patients should begin with screening of exon 3 of the BSCL2 gene. Screening of the GARS gene is useful in patients with CMT2 with predominant hand involvement and dHMN-V. The rather low frequencies of BSCL2, GARS, HSPB1 and HSPB8 mutations in dHMN-V, CMT2D and SS patients strongly point to further genetic heterogeneity of these related disorders.
Charcot–Marie–Tooth disease type 2D; CMT2; Distal hereditary motor neuropathy; dHMN; BSCL2; GARS; HSPB1; HSPB8
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.
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.
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).
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.
Considerable non-allelic heterogeneity for autosomal recessively inherited Charcot-Marie-Tooth (ARCMT) disease has challenged molecular testing and often requires a large amount of work in terms of DNA sequencing and data interpretation or remains unpractical. This study tested the value of SNP array-based whole-genome homozygosity mapping as a first step in the molecular genetic diagnosis of sporadic or ARCMT in patients from inbred families or outbred populations with the ancestors originating from the same geographic area. Using 10 K 2.0 and 250 K Nsp Affymetrix SNP arrays, 15 (63%) of 24 CMT patients received an accurate genetic diagnosis. We used our Java-based script eHoPASA CMT—easy Homozygosity Profiling of SNP arrays for CMT patients to display the location of homozygous regions and their extent of marker count and base-pairs throughout the whole genome. CMT4C was the most common genetic subtype with mutations detected in SH3TC2, one (p.E632Kfs13X) appearing to be a novel founder mutation. A sporadic patient with severe CMT was homozygous for the c.250G > C (p.G84R) HSPB1 mutation which has previously been reported to cause autosomal dominant dHMN. Two distantly related CMT1 patients with early disease onset were found to carry a novel homozygous mutation in MFN2 (p.N131S). We conclude that SNP array-based homozygosity mapping is a fast, powerful, and economic tool to guide molecular genetic testing in ARCMT and in selected sporadic CMT patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s00415-011-6213-8) contains supplementary material, which is available to authorized users.
Autosomal recessive CMT; Homozygosity mapping; SNP array; Hereditary neuropathies; Gene
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.
•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.
A large number of novel disease genes have been identified by homozygosity mapping and the positional candidate approach. In this study we used single nucleotide polymorphism (SNP) array-based, whole genome homozygosity mapping as the first step to a molecular diagnosis in the highly heterogeneous muscle disease, limb girdle muscular dystrophy (LGMD). In a consanguineous family, both affected siblings showed homozygous blocks on chromosome 15 corresponding to the LGMD2A locus. Direct sequencing of CAPN3, encoding calpain-3, identified a homozygous deletion c.483delG (p.Ile162SerfsX17). In a sporadic LGMD patient complete absence of caveolin-3 on Western blot was observed. However, a mutation in CAV3 could not be detected. Homozygosity mapping revealed a large homozygous block at the LGMD2I locus, and direct sequencing of FKRP encoding fukutin-related-protein detected the common homozygous c.826 C > A (p.Leu276Ile) mutation. Subsequent re-examination of this patient's muscle biopsy showed aberrant α-dystroglycan glycosylation. In summary, we show that whole-genome homozygosity mapping using low cost SNP arrays provides a fast and non-invasive method to identify disease-causing mutations in sporadic patients or sibs from consanguineous families in LGMD2. Furthermore, this is the first study describing that in addition to PTRF, encoding polymerase I and transcript release factor, FKRP mutations may cause secondary caveolin-3 deficiency.
CAPN3; CAV3; FKRP; Homozygosity mapping; LGMD2; SNP array
To examine insulin's effect on the tissue glucose concentration at the site of subcutaneous insulin administration.
RESEARCH DESIGN AND METHODS
A CMA-60 microdialysis (MD) catheter and a 24-gauge microperfusion (MP) catheter were inserted into the subcutaneous adipose tissue of fasting, healthy subjects (n = 5). Both catheters were perfused with regular human insulin (100 units/ml) over a 6-h period and used for glucose sampling and simultaneous administration of insulin at sequential rates of 0.33, 0.66, and 1.00 units/h (each rate was used for 2 h). Before and after the insulin delivery period, both catheters were perfused with an insulin-free solution (5% mannitol) for 2 h and used for glucose sampling only. Blood plasma glucose was clamped at euglycemic levels during insulin delivery.
Start of insulin delivery with MD and MP catheters resulted in a decline of the tissue glucose concentration and the tissue-to-plasma glucose ratio (TPR) for ∼60 min (P < 0.05). However, during the rest of the 6-h period of variable insulin delivery, tissue glucose concentration paralleled the plasma glucose concentration, and the TPR for MD and MP catheters remained unchanged at 83.2 ± 3.1 and 77.1 ± 4.8%, respectively. After subsequent switch to insulin-free perfusate, tissue glucose concentration and TPR increased slowly and reattained preinsulin delivery levels by the end of the experiments.
The results show the attainment of a stable TPR value at the site of insulin administration, thus indicating that insulin delivery and glucose sensing may be performed simultaneously at the same adipose tissue site.
Vitamin D deficiency is encountered frequently in critically ill patients and might be harmful. Current nutrition guidelines recommend very low vitamin D doses. The objective of this trial was to evaluate the safety and efficacy of a single oral high-dose vitamin D3 supplementation in an intensive care setting over a one-week observation period.
This was a randomized, double-blind, placebo-controlled pilot study in a medical ICU at a tertiary care university center in Graz, Austria. Twenty-five patients (mean age 62 ± 16yrs) with vitamin D deficiency [25-hydroxyvitamin D (25(OH)D) ≤20 ng/ml] and an expected stay in the ICU >48 hours were included and randomly received either 540,000 IU (corresponding to 13.5 mg) of cholecalciferol (VITD) dissolved in 45 ml herbal oil or matched placebo (PBO) orally or via feeding tube.
The mean serum 25(OH)D increase in the intervention group was 25 ng/ml (range 1-47 ng/ml). The highest 25(OH)D level reached was 64 ng/ml, while two patients showed a small (7 ng/ml) or no response (1 ng/ml). Hypercalcemia or hypercalciuria did not occur in any patient. From day 0 to day 7, total serum calcium levels increased by 0.10 (PBO) and 0.15 mmol/L (VITD; P < 0.05 for both), while ionized calcium levels increased by 0.11 (PBO) and 0.05 mmol/L (VITD; P < 0.05 for both). Parathyroid hormone levels decreased by 19 and 28 pg/ml (PBO and VITD, ns) over the seven days, while 1,25(OH)D showed a transient significant increase in the VITD group only.
This pilot study shows that a single oral ultra-high dose of cholecalciferol corrects vitamin D deficiency within 2 days in most patients without causing adverse effects like hypercalcemia or hypercalciuria. Further research is needed to confirm our results and establish whether vitamin D supplementation can affect the clinical outcome of vitamin D deficient critically ill patients.
German Clinical Trials Register (DRKS)
To simplify and improve the treatment of patients with type 1 diabetes, we ascertained whether the site of subcutaneous insulin infusion can be used for the measurement of glucose.
RESEARCH DESIGN AND METHODS
Three special indwelling catheters (24-gauge microperfusion [MP] catheters) were inserted into the subcutaneous adipose tissue of subjects with type 1 diabetes (n = 10; all C-peptide negative). One MP catheter was perfused with short-acting insulin (100 units/ml, Aspart) and used for insulin delivery and simultaneous glucose sampling during an overnight fast and after ingestion of a standard glucose load (75 g). As controls, the further two MP catheters were perfused with an insulin-free solution (5% mannitol) and used for glucose sampling only. Plasma glucose was measured frequently at the bedside.
Insulin delivery with the MP catheter was adequate to achieve and maintain normoglycemia during fasting and after glucose ingestion. Tissue glucose concentrations derived with the insulin-perfused catheter agreed well with plasma glucose levels. Median correlation coefficient and median absolute relative difference values were found to be 0.93 (interquartile range 0.91–0.97) and 10.9%, respectively. Error grid analysis indicated that the percentage number of tissue values falling in the clinically acceptable range is 99.6%. Comparable analysis results were obtained for the two mannitol-perfused catheters.
Our data suggest that estimation of plasma glucose concentrations from the glucose levels directly observed at the site of subcutaneous insulin infusion is feasible and its quality is comparable to that of estimating plasma glucose concentrations from glucose levels measured in insulin-unexposed subcutaneous tissue.
Intensive insulin therapy reduces mortality and morbidity in critically ill patients but places great demands on medical staff who must take frequent blood samples for the determination of glucose levels. A cost-effective solution to this resourcing problem could be provided by an effective and reliable automated blood sampling (ABS) system suitable for ex vivo glucose determination.
The primary study aim was to compare the performance of a prototype ABS system with a manual reference system over a 30 h sampling period under controlled conditions in humans. Two venous cannulae were inserted to connect the ABS system and the reference system. Blood samples were taken with both systems at 15, 30, and 60 min intervals and analyzed using a Beckman glucose analyzer. During the study, blood glucose levels were altered through four meal ingestions.
The median Pearson coefficient of correlation between manually and automatically withdrawn blood samples was 0.976 (0.953−0.996). The system error was −3.327 ± 5.546% (−6.03−0.49). Through Clark error grid analysis, 420 data pairs were analyzed, showing that 98.6% of the data were in zone A and 1.4% were in zone B. Insulin titration error grid analysis revealed an acceptable treatment in 100% of cases. A 17.5-fold reduction in the occurrence of blood-withdrawal failures through occluded catheters was moreover achieved by the added implementation in the ABS system of a “keep vein open” saline infusion.
Our study showed that the ABS system described provides a user-friendly, reliable automated means for reproducible and accurate blood sampling from a peripheral vein for blood glucose determination and thus represents a promising alternative to frequent manual blood sampling.
automated; blood; glucose; monitoring; sampling; ex vivo