Normative data on measures of cardiovascular autonomic neuropathy and the effect of pretest conditions in a large Danish non-diabetic CVD-free population from the Lolland-Falster Health Study.

PURPOSE: Cardiovascular autonomic neuropathy (CAN) is a common diabetic complication associated with excess morbidity and mortality. CAN is also seen in conditions such as Parkinson's disease. Normative reference data for cardiovascular autonomic function are used to stratify individuals into those with and without CAN. However, reference thresholds for both cardiovascular autonomic reflex tests (CARTs) and heart rate variability (HRV) are scarce and based on small sample sizes. The aim of the study was to establish contemporary normative reference thresholds based on a large non-diabetic population free of cardiovascular disease (CVD). METHODS: Cardiovascular autonomic function, CARTs and 5-min HRV indices were assessed in individuals without diabetes and CVD from the Lolland-Falster Health Study (2018-2020) by applying the point-of-care device Vagus™. Age-specific normative reference thresholds were estimated by using log-transformed quantile regression models at the 5th and 10th percentile, with adjustments made for sex. Models assessing the association between age and HRV indices were further adjusted for heart rate. RESULTS: We present age-specific normative reference thresholds for cardiovascular autonomic function, including CARTs and HRV, for 875 individuals (48% females) aged 15-85 years. The reference thresholds are presented for both the 5th and 10th lower percentile. Higher age was inversely associated with all outcomes. Females tended to have a higher parasympathetic drive compared to males. Pre-test conditions did not affect CARTs significantly. CONCLUSIONS: The presented age-related normative reference thresholds for both CARTs and HRV indices based on a large Danish cohort may facilitate improved quality of research and treatment.

Transcutaneous vagal nerve stimulation for treating gastrointestinal symptoms in individuals with diabetes: a randomised, double-blind, sham-controlled, multicentre trial.

AIMS/HYPOTHESIS: Diabetic gastroenteropathy frequently causes debilitating gastrointestinal symptoms. Previous uncontrolled studies have shown that transcutaneous vagal nerve stimulation (tVNS) may improve gastrointestinal symptoms. To investigate the effect of cervical tVNS in individuals with diabetes suffering from autonomic neuropathy and gastrointestinal symptoms, we conducted a randomised, sham-controlled, double-blind (participants and investigators were blinded to the allocated treatment) study. METHODS: This study included adults (aged 20-86) with type 1 or 2 diabetes, gastrointestinal symptoms and autonomic neuropathy recruited from three Steno Diabetes Centres in Denmark. Participants were randomly allocated 1:1 to receive active or sham stimulation. Active cervical tVNS or sham stimulation was self-administered over two successive study periods: 1 week of four daily stimulations and 8 weeks of two daily stimulations. The primary outcome measures were gastrointestinal symptom changes as measured using the gastroparesis cardinal symptom index (GCSI) and the gastrointestinal symptom rating scale (GSRS). Secondary outcomes included gastrointestinal transit times and cardiovascular autonomic function. RESULTS: Sixty-eight participants were randomised to the active group, while 77 were randomised to the sham group. Sixty-three in the active and 68 in the sham group remained for analysis in study period 1, while 62 in each group were analysed in study period 2. In study period 1, active and sham tVNS resulted in similar symptom reductions (GCSI: -0.26 ± 0.64 vs -0.17 ± 0.62, p=0.44; GSRS: -0.35 ± 0.62 vs -0.32 ± 0.59, p=0.77; mean ± SD). In study period 2, active stimulation also caused a mean symptom decrease that was comparable to that observed after sham stimulation (GCSI: -0.47 ± 0.78 vs -0.33 ± 0.75, p=0.34; GSRS: -0.46 ± 0.90 vs -0.35 ± 0.79, p=0.50). Gastric emptying time was increased in the active group compared with sham (23 min vs -19 min, p=0.04). Segmental intestinal transit times and cardiovascular autonomic measurements did not differ between treatment groups (all p>0.05). The tVNS was well-tolerated. CONCLUSIONS/INTERPRETATION: Cervical tVNS, compared with sham stimulation, does not improve gastrointestinal symptoms among individuals with diabetes and autonomic neuropathy. TRIAL REGISTRATION: ClinicalTrials.gov NCT04143269 FUNDING: The study was funded by the Novo Nordisk Foundation (grant number NNF180C0052045).

Structural Changes of Cutaneous Immune Cells in Patients With Type 1 Diabetes and Their Relationship With Diabetic Polyneuropathy.

BACKGROUND AND OBJECTIVES: Diabetic polyneuropathy (DPN) is a complication of diabetes characterized by pain or lack of peripheral sensation, but the underlying mechanisms are not yet fully understood. Recent evidence showed increased cutaneous macrophage infiltration in patients with type 2 diabetes and painful DPN, and this study aimed to understand whether the same applies to type 1 diabetes. METHODS: The study included 104 participants: 26 healthy controls and 78 participants with type 1 diabetes (participants without DPN [n = 24], participants with painless DPN [n = 29], and participants with painful DPN [n = 25]). Two immune cells, dermal IBA1+ macrophages and epidermal Langerhans cells (LCs, CD207+), were visualized and quantified using immunohistological labeling and stereological counting methods on skin biopsies from the participants. The IBA1+ macrophage infiltration, LC number density, LC soma cross-sectional area, and LC processes were measured in this study. RESULTS: Significant difference in IBA1+ macrophage expression was seen between the groups (p = 0.003), with lower expression of IBA1 in participants with DPN. No differences in LC morphologies (LC number density, soma cross-sectional area, and process level) were found between the groups (all p > 0.05). In addition, IBA1+ macrophages, but not LCs, correlated with intraepidermal nerve fiber density, Michigan neuropathy symptom inventory, (questionnaire and total score), severity of neuropathy as assessed by the Toronto clinical neuropathy score, and vibration detection threshold in the whole study cohort. DISCUSSION: This study showed expressional differences of cutaneous IBA1+ macrophages but not LC in participants with type 1 diabetes-induced DPN compared with those in controls. The study suggests that a reduction in macrophages may play a role in the development and progression of autoimmune-induced diabetic neuropathy.

Collagen turnover is associated with cardiovascular autonomic and peripheral neuropathy in type 1 diabetes: novel pathophysiological mechanism?

BACKGROUND: Diabetic cardiovascular autonomic neuropathy (CAN) and distal symmetrical polyneuropathy (DSPN) are severe diabetic complications. Collagen type VI (COL6) and III (COL3) have been associated with nerve function. We investigated if markers of COL6 formation (PRO-C6) and COL3 degradation (C3M) were associated with neuropathy in people with type 1 diabetes (T1D). METHODS: In a cross-sectional study including 300 people with T1D, serum and urine PRO-C6 and C3M were obtained. CAN was assessed by cardiovascular reflex tests: heart rate response to deep breathing (E/I ratio), to standing (30/15 ratio) and to the Valsalva maneuver (VM). Two or three pathological CARTs constituted CAN. DSPN was assessed by biothesiometry. Symmetrical vibration sensation threshold above 25 V constituted DSPN. RESULTS: Participants were (mean (SD)) 55.7 (9.3) years, 51% were males, diabetes duration was 40.0 (8.9) years, HbA1c was 63 (11 mmol/mol, (median (IQR)) serum PRO-C6 was 7.8 (6.2;11.0) ng/ml and C3M 8.3 (7.1;10.0) ng/ml. CAN and DSPN were diagnosed in 34% and 43% of participants, respectively. In models adjusted for relevant confounders a doubling of serum PRO-C6, was significantly associated with odds ratio > 2 for CAN and > 1 for DSPN, respectively. Significance was retained after additional adjustments for eGFR only for CAN. Higher serum C3M was associated with presence of CAN, but not after adjustment for eGFR. C3M was not associated with DSPN. Urine PRO-C6 analyses indicated similar associations. CONCLUSIONS: Results show previously undescribed associations between markers of collagen turnover and risk of CAN and to a lesser degree DSPN in T1D.

Autonomic dysfunction is associated with the development of arterial stiffness: the Whitehall II cohort.

This study aims to examine the association between baseline level and change of autonomic nervous function with subsequent development of arterial stiffness. Autonomic nervous function was assessed in 4901 participants of the Whitehall II occupational cohort by heart rate variability (HRV) indices and resting heart rate (rHR) three times between 1997 and 2009, while arterial stiffness was assessed by carotid-femoral pulse wave velocity (PWV) measured twice between 2007 and 2013. First, individual HRV/rHR levels and annual changes were estimated. Then, we modelled the development of PWV by HRV/rHR using linear mixed effect models. First, we adjusted for sex and ethnicity (model 1), and then for socioeconomic and lifestyle factors, various clinical measurements, and medications (model 2). A decrease in HRV and unchanged rHR was associated with subsequent higher levels of PWV, but the effect of a change in HRV was less pronounced at higher ages. A typical individual aged 65 years with a SDNN level of 30 ms and a 2% annual decrease in SDNN had 1.32 (0.95; 1.69) higher PWV compared to one with the same age and SDNN level but with a 1% annual decrease in SDNN. Further adjustment had no major effect on the results. People who experience a steeper decline in autonomic nervous function have higher levels of arterial stiffness. The association was stronger in younger people.

Advanced glycation end-products are associated with diabetic neuropathy in young adults with type 1 diabetes.

Aims/hypothesis: Advanced glycation end-products (AGEs) may contribute to the development of diabetic neuropathy. In young adults with type 1 diabetes, we aimed to investigate the association between AGEs and cardiovascular autonomic neuropathy (CAN) and distal symmetric polyneuropathy (DSPN). Methods: This cross-sectional study comprised 151 young adults. CAN was assessed by cardiovascular autonomic reflex tests; lying-to-standing test, deep breathing test (E/I), Valsalva manoeuvre, and heart rate variability indices; and the mean square of the sum of the squares of differences between consecutive R-R intervals and standard deviation of normal-to-normal intervals (SDNN), high- (HF) and low-frequency (LF) power, total frequency power, and the LF/HF ratio. DSPN was assessed by light touch, pain and vibration perception threshold (VPT), neuropathy questionnaires, and objective measures. AGEs were analysed in four groups using z-scores adjusted for relevant confounders and multiple testing: i) "glycolytic dysfunction", ii) "lipid peroxidation", iii) "oxidative stress", and iv) "glucotoxicity". Results: A higher z-score of "glycolytic dysfunction" was associated with higher VPT (4.14% (95% CI 1.31; 7.04), p = 0.004) and E/I (0.03% (95% CI 0.01; 0.05), p = 0.005), "lipid peroxidation" was associated with higher LF/HF ratio (37.72% (95% CI 1.12; 87.57), p = 0.044), and "glucotoxicity" was associated with lower SDNN (-4.20% (95% CI -8.1416; -0.0896), p = 0.047). No significance remained after adjustment for multiple testing. Conclusions/interpretations: In young adults with type 1 diabetes, increased levels of AGEs involving different metabolic pathways were associated with several measures of CAN and DSPN, suggesting that AGEs may play a diverse role in the pathogeneses of diabetic neuropathy.

Cardiovascular Autonomic Neuropathy Is Associated With Increased Glucose Variability in People With Type 1 Diabetes.

OBJECTIVE: We investigated the association between the cardiovascular autonomic neuropathy (CAN) diagnosis and glucose variability (GV) in type 1 diabetes (T1D), as autonomic dysfunction previously has been associated with increased GV. RESEARCH DESIGN AND METHODS: CAN was assessed by three recommended cardiovascular reflex tests (CARTs). Glucose metrics were obtained from 10-day blinded continuous glucose monitoring (CGM). Between-group differences in GV indices were assessed by regression analyses in 24 participants with T1D with CAN and 24 matched control subjects without CAN. RESULTS: The CAN diagnosis was associated with 4.9% (95% CI 1.0, 8.7) higher coefficient of variation (CV) (P = 0.014), 0.7 mmol/L (0.3, 1.1) higher SD (P = 0.002) of glucose, and 1.4 mmol/mol (0.0, 2.7) higher mean amplitude of glycemic excursions (P = 0.047). Lower measures of CARTs were associated with higher CV, SD, and time above range values. CONCLUSIONS: The CAN diagnosis associates with a significantly higher GV in T1D, despite a high prevalence of routine CGM use.

Cardiovascular Autonomic Neuropathy in Type 1 Diabetes Is Associated With Disturbances in TCA, Lipid, and Glucose Metabolism.

Introduction: Diabetic cardiovascular autonomic neuropathy (CAN) is associated with increased mortality and morbidity. To explore metabolic mechanisms associated with CAN we investigated associations between serum metabolites and CAN in persons with type 1 diabetes (T1D). Materials and Methods: Cardiovascular reflex tests (CARTs) (heart rate response to: deep breathing; lying-to-standing test; and the Valsalva maneuver) were used to diagnose CAN in 302 persons with T1D. More than one pathological CARTs defined the CAN diagnosis. Serum metabolomics and lipidomic profiles were analyzed with two complementary non-targeted mass-spectrometry methods. Cross-sectional associations between metabolites and CAN were assessed by linear regression models adjusted for relevant confounders. Results: Participants were median (IQR) aged 55(49, 63) years, 48% males with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had CAN. A total of 75 metabolites and 106 lipids were analyzed. In crude models, the CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4-deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and the amino acid methionine (p<0.05). Upon adjustment, positive associations with the CAN diagnoses were retained for hydroxy fatty acids, tricarboxylic acid (TCA) cycle-based sugar derivates, citric acid, and phenols (P<0.05). Conclusion: Metabolic pathways, including the TCA cycle, hydroxy fatty acids, phosphatidylcholines and sugar derivatives are associated with the CAN diagnosis in T1D. These pathway may be part of the pathogeneses leading to CAN and may be modifiable risk factors for the complication.

The Association Between Cardiovascular Autonomic Function and Changes in Kidney and Myocardial Function in Type 2 Diabetes and Healthy Controls.

Background: The mechanisms linking cardiovascular autonomic neuropathy, diabetic kidney disease and cardiovascular mortality in type 2 diabetes are widely unknown. We investigated the relationship between baseline cardiovascular autonomic function and changes in kidney and myocardial function over six years in patients with type 2 diabetes and healthy controls. Methods: Post-hoc analysis of a cohort study in 24 patients with type 2 diabetes and 18 healthy controls. Baseline determinants were cardiovascular autonomic reflex tests (heart rate response to: standing (30:15); deep breathing (E:I); and the Valsalva test) and time- and frequency-domain heart rate variability indices. Outcomes were changes in estimated glomerular filtration rate (eGFR), albuminuria, myocardial flow reserve (MFR) measured by cardiac 82Rb Positron emission tomography computed tomography (PET/CT), and coronary artery calcium score (CACS). Results: Mean age at inclusion was 61 ± 10 years and 36% were female. Mean follow up time was 6 ± 0 years. A lower response in heart rate to the Valsalva test (corresponding to weaker autonomic function) was associated with a larger decline in eGFR (p=0.04), but not significantly after adjustment for sex, baseline age, smoking status, systolic blood pressure, heart rate, HbA1c, body mass index and baseline eGFR (p=0.12). A higher baseline response in heart rate to standing (30:15) was associated with a larger decline in myocardial flow reserve in the unadjusted analysis (p=0.02) and after adjustment (p=0.02). A higher response in heart rate to the Valsalva maneuver was associated with a larger increase in CACS (p = 0.02), but the association became insignificant after adjustment (p = 0.16). Conclusion: A lower response in heart rate to the Valsalva test was associated with a larger decline in kidney function, indicating that autonomic dysfunction may predict future loss of kidney function. However, we did not find any association between lower values in cardiovascular autonomic function at baseline and a worsening in albuminuria, myocardial function, or atherosclerotic burden.

Heart Rate and Heart Rate Variability Changes Are Not Related to Future Cardiovascular Disease and Death in People With and Without Dysglycemia: A Downfall of Risk Markers? The Whitehall II Cohort Study.

OBJECTIVE: Higher resting heart rate (rHR) and lower heart rate variability (HRV) are associated with increased risk of cardiovascular disease (CVD) and all-cause mortality in people with and without diabetes. It is unknown whether temporal changes in rHR and HRV may contribute to this risk. We investigated associations between 5-year changes in rHR and HRV and risk of future CVD and death, taking into account participants' baseline glycemic state. RESEARCH DESIGN AND METHODS: In this prospective, population-based cohort study we investigated 4,611 CVD-free civil servants (mean [SD] age, 60 [5.9] years; 70% men). We measured rHR and/or six indices of HRV. Associations of 5-year change in 5-min rHR and HRV with fatal and nonfatal CVD and all-cause mortality or the composite of the two were assessed, with adjustments made for relevant confounders. Effect modification by glycemic state was tested. RESULTS: At baseline, 63% of participants were normoglycemic, 29% had prediabetes, and 8% had diabetes. During a median (interquartile range) follow-up of 11.9 (11.4; 12.3) years, 298 participants (6.5%) experienced a CVD event and 279 (6.1%) died of non-CVD-related causes. We found no association between 5-year changes in rHR and HRV and future events. Only baseline rHR was associated with all-cause mortality. A 10 bpm-higher baseline HR level was associated with an 11.4% higher rate of all-cause mortality (95% CI 1.0-22.9%; P = 0.032). Glycemic state did not modify associations. CONCLUSIONS: Changes in rHR and HRV and possibly also baseline values of these measures are not associated with future CVD or death in people with or without dysglycemia.

Epicardial adipose tissue predicts incident cardiovascular disease and mortality in patients with type 2 diabetes.

BACKGROUND: Cardiac fat is a cardiovascular biomarker but its importance in patients with type 2 diabetes is not clear. The aim was to evaluate the predictive potential of epicardial (EAT), pericardial (PAT) and total cardiac (CAT) fat in type 2 diabetes and elucidate sex differences. METHODS: EAT and PAT were measured by echocardiography in 1030 patients with type 2 diabetes. Follow-up was performed through national registries. The end-point was the composite of incident cardiovascular disease (CVD) and all-cause mortality. Analyses were unadjusted (model 1), adjusted for age and sex (model 2), plus systolic blood pressure, body mass index (BMI), low-density lipoprotein (LDL), smoking, diabetes duration and glycated hemoglobin (HbA1c) (model 3). RESULTS: Median follow-up was 4.7 years and 248 patients (191 men vs. 57 women) experienced the composite end-point. Patients with high EAT (> median level) had increased risk of the composite end-point in model 1 [Hazard ratio (HR): 1.46 (1.13; 1.88), p = 0.004], model 2 [HR: 1.31 (1.01; 1.69), p = 0.038], and borderline in model 3 [HR: 1.32 (0.99; 1.77), p = 0.058]. For men, but not women, high EAT was associated with a 41% increased risk of CVD and mortality in model 3 (p = 0.041). Net reclassification index improved when high EAT was added to model 3 (19.6%, p = 0.035). PAT or CAT were not associated with the end-point. CONCLUSION: High levels of EAT were associated with the composite of incident CVD and mortality in patients with type 2 diabetes, particularly in men, after adjusting for CVD risk factors. EAT modestly improved risk prediction over CVD risk factors.

Efficacy of Long-Term Remote Ischemic Conditioning on Vascular and Neuronal Function in Type 2 Diabetes Patients With Peripheral Arterial Disease.

Background Peripheral artery disease is a major socioeconomic challenge in the diabetes mellitus community and non-surgical treatment options are limited. As remote ischemic conditioning ( RIC ) improves vascular function and attenuates ischemia-induced tissue damage, we investigated the efficacy of RIC on vascular and neuronal function in type 2 diabetes mellitus patients with peripheral artery disease. Methods and Results We enrolled 36 type 2 diabetes mellitus patients with moderately reduced toe pressure (40-70 mm Hg) in a randomized sham-controlled double-masked trial. Patients were allocated to 12 weeks once daily upper arm cuff-based treatment of either RIC treatment (4 cycles of 5-minute ischemia followed by 5-minute reperfusion) or similar sham-device treatment. Primary outcome was transcutaneous tissue oxygen tension of the instep of the feet. Secondary outcomes were aortic pulse wave velocity, toe pressure and toe-brachial index. Tertiary outcomes were markers of peripheral and autonomic nerve function. We enrolled 36 patients (83% men). Patients had a mean ( SD ) age of 70.7 years (6.8), diabetes mellitus duration of 18.4 years (8.3), HbA1c (gycated hemoglobin) of 59.7 mmol/mol (11.2). Eighty percent had peripheral symmetrical neuropathy. The mean difference in change of transcutaneous tissue oxygen tension from baseline between the RIC and sham-treated groups was -0.03 mm Hg ([95% CI -0.1; 0.04], P=0.438). RIC did not elicit any change in additional outcomes. Three patients experienced transient skin petechiae in the treated arm. Conclusions Long-term repeated remote ischemic conditioning treatment have no effect on tissue oxygenation, vascular or neuronal function in patients with type 2 diabetes mellitus and moderate peripheral artery disease. Clinical Trial Registration URL : http://www.ClinicalTrials.gov . Unique identifier: NCT02749942.

Epicardial and pericardial adipose tissues are associated with reduced diastolic and systolic function in type 2 diabetes.

The aim of this study was to investigate the association of epicardial (EAT) and pericardial (PAT) adipose tissues with myocardial function in type 2 diabetes (T2D). EAT and PAT were measured by ultrasound in 770 patients with T2D and 234 age- and sex-matched non-diabetic controls. Echocardiography was performed, including tissue Doppler imaging and 2D speckle tracking. Patients with T2D versus controls had increased EAT (4.6 ± 1.8 mm vs. 3.4 ± 1.2 mm, P < 0.0001) and PAT (6.3 ± 2.8 mm vs. 5.3 ± 2.4 mm, P < 0.0001). EAT and PAT were associated with structural cardiac measures both in T2D patients and controls (all P < 0.043), but only in T2D patients with functional measures: PAT was associated with impaired global longitudinal strain [beta coefficient (SE)] [0.11% (0.04), P = 0.002], while EAT was associated with reduced diastolic function by lateral early diastolic myocardial velocity (e'lat ) [-0.31 (0.05) cm/s, P = 0.001], mitral inflow velocities: peak early (E)/peak atrial (A) ratio [-0.02 (0.01), P = 0.001] and lateral E/e'lat [0.36 (0.10), P < 0.001]. However, no interaction was found between diabetes status and PAT (P = 0.75) or EAT (P = 0.45). Adipose tissue in intimate relation to the myocardium is higher in patients with T2D versus controls and is associated with functional myocardial measures in T2D.

Epicardial, pericardial and total cardiac fat and cardiovascular disease in type 2 diabetic patients with elevated urinary albumin excretion rate.

Background We evaluated the association of cardiac adipose tissue including epicardial adipose tissue and pericardial adipose tissue with incident cardiovascular disease and mortality, coronary artery calcium, carotid intima media thickness and inflammatory markers. Design A prospective study of 200 patients with type 2 diabetes and elevated urinary albumin excretion rate (UAER). Methods Cardiac adipose tissue was measured from baseline echocardiography. The composite endpoint comprised incident cardiovascular disease and all-cause mortality. Coronary artery calcium, carotid intima media thickness and inflammatory markers were measured at baseline. Cardiac adipose tissue was investigated as continuous and binary variable. Analyses were performed unadjusted (model 1), and adjusted for age, sex (model 2), body mass index, low-density lipoprotein cholesterol, smoking, glycated haemoglobin, and systolic blood pressure (model 3). Results Patients were followed-up after 6.1 years for non-fatal cardiovascular disease ( n = 29) or mortality ( n = 23). Cardiac adipose tissue ( p = 0.049) and epicardial adipose tissue ( p = 0.029) were associated with cardiovascular disease and mortality in model 1. When split by the median, patients with high cardiac adipose tissue had a higher risk of cardiovascular disease and mortality than patients with low cardiac adipose tissue in unadjusted (hazard ratio 1.9, confidence interval: 1.1; 3.4, p = 0.027) and adjusted (hazard ratio 2.0, confidence interval: 1.1; 3.7, p = 0.017) models. Cardiac adipose tissue ( p = 0.033) was associated with baseline coronary artery calcium (model 1) and interleukin-8 (models 1-3, all p < 0.039). Conclusions In type 2 diabetes patients without coronary artery disease, high cardiac adipose tissue levels were associated with increased risk of incident cardiovascular disease or all-cause mortality even after accounting for traditional cardiovascular disease risk factors. High cardiac adipose tissue amounts were associated with subclinical atherosclerosis (coronary artery calcium) and with the pro-atherogenic inflammatory marker interleukin-8.

Vitamin B12 deficiency is associated with cardiovascular autonomic neuropathy in patients with type 2 diabetes.

AIMS: Vitamin B12 deficiency could be associated with cardiovascular autonomic neuropathy (CAN) in diabetes patients. We aim to investigate the association between serum levels of vitamin B12 and CAN in type 2 diabetes patients. METHODS: 469 ambulatory type 2 diabetes patients (mean diabetes duration 10.0years (IQR 5.0;17.0), mean age 59.0years (SD 11.6), 63% men, mean B12 289.0pmol/l (IQR 217;390)) were screened for CAN using three cardiovascular reflex tests, five minute resting heart rate (5min RHR) and heart rate variability indices. RESULTS: Serum levels of vitamin B12 were significantly lower in patients treated with metformin and/or proton pump inhibitors (PPIs) compared with patients not treated (p<0.001). A 25pmol/l higher level of vitamin B12 was associated with an odds ratio of the CAN diagnosis of 0.94 (95% CI 0.88; 1.00, p=0.034), an increase in E/I-ratio of 0.21% (95% CI 0.01; 0.43, p=0.038), and a decrease in 5min RHR of 0.25 beats per minute (95% CI -0.47; -0.03, p=0.025). CONCLUSION: Vitamin B12 may be inversely associated with CAN in patients with type 2 diabetes. Confirmatory studies investigating a causal role of vitamin B12 for the development of diabetic CAN are warranted.

High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays.

Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated.

Making sense of a new technology in clinical practice: a qualitative study of patient and physician perspectives.

BACKGROUND: The number of new technologies for risk assessment available in health care is increasing. These technologies are intended to contribute to both improved care practices and improved patient outcomes. To do so however, there is a need to study how new technologies are understood and interpreted by users in clinical practice. The objective of this study was to explore patient and physician perspectives on the usefulness of a new technology to detect Cardiovascular Autonomic Neuropathy (CAN) in a specialist diabetes clinic. The technology is a handheld device that measures resting heart rate and conducts three cardiac autonomic reflex tests to evaluate heart rate variability. METHODS: The study relied on three sources of data: observations of medical consultations where results of the CAN test were reported (n = 8); interviews with patients who had received the CAN test (n = 19); and interviews with physicians who reported results of the CAN test (n = 9). Data were collected at the specialist diabetes clinic between November 2013 and January 2014. Data were analysed using the concept of technological frames which is used to assess how physicians and patients understand and interpret the new technology. RESULTS: Physicians generally found it difficult to communicate test results to patients in terms that patients could understand and to translate results into meaningful implications for the treatment of patients. Results of the study indicate that patients did not recall having done the CAN test nor recall receiving the results. Furthermore, patients were generally unsure about the purpose of the CAN test and the implications of the results. DISCUSSION: Involving patients and physicians is essential when a new technology is introduced in clinical practice. This particularly includes the interpretation and communication processes related to its use. CONCLUSIONS: The integration of a new risk assessment technology into clinical practice can be accompanied by several challenges. It is suggested that more information about the CAN test be provided to patients and that a dialogue-based approach be used when communicating test results to patients in order to best support the use of the technology in clinical practice.

High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum.

Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds.