Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process

Sympathetic Activation and Baroreflex Function during Intradialytic Hypertensive Episodes

BACKGROUND: The mechanisms of intradialytic increases in blood pressure are not well defined. The present study was undertaken to assess the role of autonomic nervous system activation during intradialytic hypertensive episodes. METHODOLOGY/PRINCIPAL FINDINGS: Continuous interbeat intervals (IBI) and systolic blood pressure (SBP) were monitored during hemodialysis in 108 chronic patients. Intradialytic hypertensive episodes defined as a period of at least 10 mmHg increase in SBP between the beginning and the end of a dialysis session or hypertension resistant to ultrafiltration occurring during or immediately after the dialysis procedure, were detected in 62 out of 113 hemodialysis sessions. SBP variability, IBI variability and baroreceptor sensitivity (BRS) in the low (LF) and high (HF) frequency ranges were assessed using the complex demodulation technique (CDM). Intradialytic hypertensive episodes were associated with an increased (n = 45) or decreased (n = 17) heart rate. The maximal blood pressure was similar in both groups. In patients with increased heart rate the increase in blood pressure was associated with marked increases in SBP and IBI variability, with suppressed BRS indices and enhanced sympatho-vagal balance. In contrast, in those with decreased heart rate, there were no significant changes in the above parameters. End-of-dialysis blood pressure in all sessions associated with hypertensive episode was significantly higher than in those without such episodes. In logistic regression analysis, predialysis BRS in the low frequency range was found to be the main predictor of intradialytic hypertension. CONCLUSION/SIGNIFICANCE: Our data point to sympathetic overactivity with feed-forward blood pressure enhancement as an important mechanism of intradialytic hypertension in a significant proportion of patients. The triggers of increased sympathetic activity during hemodialysis remain to be determined. Intradialytic hypertensive episodes are associated with higher end-of-dialysis blood pressure, suggesting that intradialytic hypertension may play a role in generation of interdialytic hypertension

Acute mountain sickness.

Acute mountain sickness (AMS) is a clinical syndrome occurring in otherwise healthy normal individuals who ascend rapidly to high altitude. Symptoms develop over a period ofa few hours or days. The usual symptoms include headache, anorexia, nausea, vomiting, lethargy, unsteadiness of gait, undue dyspnoea on moderate exertion and interrupted sleep. AMS is unrelated to physical fitness, sex or age except that young children over two years of age are unduly susceptible. One of the striking features ofAMS is the wide variation in individual susceptibility which is to some extent consistent. Some subjects never experience symptoms at any altitude while others have repeated attacks on ascending to quite modest altitudes. Rapid ascent to altitudes of 2500 to 3000m will produce symptoms in some subjects while after ascent over 23 days to 5000m most subjects will be affected, some to a marked degree. In general, the more rapid the ascent, the higher the altitude reached and the greater the physical exertion involved, the more severe AMS will be. Ifthe subjects stay at the altitude reached there is a tendency for acclimatization to occur and symptoms to remit over 1-7 days

Effect of sitagliptin on cardiovascular outcomes in type 2 diabetes

BACKGROUND: Data are lacking on the long-term effect on cardiovascular events of adding sitagliptin, a dipeptidyl peptidase 4 inhibitor, to usual care in patients with type 2 diabetes and cardiovascular disease. METHODS: In this randomized, double-blind study, we assigned 14,671 patients to add either sitagliptin or placebo to their existing therapy. Open-label use of antihyperglycemic therapy was encouraged as required, aimed at reaching individually appropriate glycemic targets in all patients. To determine whether sitagliptin was noninferior to placebo, we used a relative risk of 1.3 as the marginal upper boundary. The primary cardiovascular outcome was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. RESULTS: During a median follow-up of 3.0 years, there was a small difference in glycated hemoglobin levels (least-squares mean difference for sitagliptin vs. placebo, -0.29 percentage points; 95% confidence interval [CI], -0.32 to -0.27). Overall, the primary outcome occurred in 839 patients in the sitagliptin group (11.4%; 4.06 per 100 person-years) and 851 patients in the placebo group (11.6%; 4.17 per 100 person-years). Sitagliptin was noninferior to placebo for the primary composite cardiovascular outcome (hazard ratio, 0.98; 95% CI, 0.88 to 1.09; P<0.001). Rates of hospitalization for heart failure did not differ between the two groups (hazard ratio, 1.00; 95% CI, 0.83 to 1.20; P = 0.98). There were no significant between-group differences in rates of acute pancreatitis (P = 0.07) or pancreatic cancer (P = 0.32). CONCLUSIONS: Among patients with type 2 diabetes and established cardiovascular disease, adding sitagliptin to usual care did not appear to increase the risk of major adverse cardiovascular events, hospitalization for heart failure, or other adverse events

Effects of Once-Weekly Exenatide on Cardiovascular Outcomes in Type 2 Diabetes.

Abstract BACKGROUND: The cardiovascular effects of adding once-weekly treatment with exenatide to usual care in patients with type 2 diabetes are unknown. METHODS: We randomly assigned patients with type 2 diabetes, with or without previous cardiovascular disease, to receive subcutaneous injections of extended-release exenatide at a dose of 2 mg or matching placebo once weekly. The primary composite outcome was the first occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. The coprimary hypotheses were that exenatide, administered once weekly, would be noninferior to placebo with respect to safety and superior to placebo with respect to efficacy. RESULTS: In all, 14,752 patients (of whom 10,782 [73.1%] had previous cardiovascular disease) were followed for a median of 3.2 years (interquartile range, 2.2 to 4.4). A primary composite outcome event occurred in 839 of 7356 patients (11.4%; 3.7 events per 100 person-years) in the exenatide group and in 905 of 7396 patients (12.2%; 4.0 events per 100 person-years) in the placebo group (hazard ratio, 0.91; 95% confidence interval [CI], 0.83 to 1.00), with the intention-to-treat analysis indicating that exenatide, administered once weekly, was noninferior to placebo with respect to safety (P<0.001 for noninferiority) but was not superior to placebo with respect to efficacy (P=0.06 for superiority). The rates of death from cardiovascular causes, fatal or nonfatal myocardial infarction, fatal or nonfatal stroke, hospitalization for heart failure, and hospitalization for acute coronary syndrome, and the incidence of acute pancreatitis, pancreatic cancer, medullary thyroid carcinoma, and serious adverse events did not differ significantly between the two groups. CONCLUSIONS: Among patients with type 2 diabetes with or without previous cardiovascular disease, the incidence of major adverse cardiovascular events did not differ significantly between patients who received exenatide and those who received placebo. (Funded by Amylin Pharmaceuticals; EXSCEL ClinicalTrials.gov number, NCT01144338 .)

Association of change in brain structure to objectively measured physical activity and sedentary behavior in older adults: Age, Gene/Environment Susceptibility-Reykjavik Study

Many studies have examined the hypothesis that greater participation in physical activity (PA) is associated with less brain atrophy. Here we examine, in a sub-sample (n = 352, mean age 79.1 years) of the Age, Gene/Environment Susceptibility-Reykjavik Study cohort, the association of the baseline and 5-year change in magnetic resonance imaging (MRI)-derived volumes of gray matter (GM) and white matter (WM) to active and sedentary behavior (SB) measured at the end of the 5-year period by a hip-worn accelerometer for seven consecutive days. More GM (beta = 0.11; p = 0.044) and WM (beta = 0.11; p = 0.030) at baseline was associated with more total physical activity (TPA). Also, when adjusting for baseline values, the 5-year change in GM (beta = 0.14; p = 0.0037) and WM (beta = 0.11; p = 0.030) was associated with TPA. The 5-year change in WM was associated with SB (p = 0.11; p = 0.0007). These data suggest that objectively measured PA and SB late in life are associated with current and prior cross-sectional measures of brain atrophy, and that change over time is associated with PA and SB in expected directions