Gel-type autologous chondrocyte implantation for cartilage repair in patients with prior ACL reconstruction: A retrospective two year follow-up.

PURPOSE: To describe the early patient-reported outcomes of articular cartilage repair in patients with pain due to grade III or IV articular cartilage defects after prior anterior cruciate ligament (ACL) reconstruction. METHODS: Nineteen patients underwent a gel-type autologous chondrocyte implantation (GACI) procedure after ACL reconstruction. Median timeframe between ACL reconstruction and GACI procedure was 52 months (range 16 to 369). The average age at chondrocyte implantation was 35 (standard deviation (SD) eight) years and average cumulative articular cartilage defect size was nine (SD four) square centimeter. Outcome was assessed prior to the GACI procedure and two years after GACI using the International Knee Documentation Committee (IKDC) score and the Knee injury and Osteoarthritis Outcome Score (KOOS). RESULTS: Two year post-GACI scores showed a statistically significant improvement of IKDC (13, SD 22, p=.02) and KOOS quality of life (18, SD 27, p=.01) compared to the pre-GACI scores. The other KOOS domains did improve, but not statistically significant. Seven (37%) patients underwent reoperation after the GACI. CONCLUSION: Patients with prior ACL reconstruction and suffering from ongoing pain associated with cartilage defects can benefit from cartilage repair with GACI.

Determination of dabigatran and rivaroxaban by ultra-performance liquid chromatography-tandem mass spectrometry and coagulation assays after major orthopaedic surgery.

Major orthopaedic surgery is associated with an increased risk of venous thromboembolism. Direct oral anticoagulants (DOACs) are recommended as thromboprophylactic agents after orthopaedic surgery. Although routine monitoring of DOACs in general is not required, measuring DOAC concentration may be necessary in clinical settings. The effects of DOACs on routine coagulation assays in spiked material are studied extensively, however, few data are available on DOAC concentration in patients after major orthopaedic surgery. We measured trough and peak DOAC concentrations with UPLC-MS/MS and routine coagulation tests in a prospective study including 40 patients receiving thromboprophylactic treatment with dabigatran 220mg od and 40 patients receiving rivaroxaban 10mg od after major orthopaedic surgery. For rivaroxaban, the median trough concentration with UPLC-MS/MS was 17.1ng/mL and median peak concentration was 149ng/mL. The anti-Xa assay displayed a good correlation, but a positive bias in comparison to the reference method. Furthermore, trough levels were mostly below the LOD of the anti-Xa assay. For dabigatran, the median trough concentration with UPLC-MS/MS was 12.1ng/mL, and median peak level was 80.8ng/mL. A positive bias was found when results from coagulation assays were compared to UPLC-MS/MS data. However, the addition of glucuronidated metabolites to dabigatran concentration UPLC-MS/MS data generally resolved most of this bias. Age was found to have a significant influence on dabigatran pharmacokinetics, irrespective of kidney function, whereas no effect of age was found during rivaroxaban treatment. In both treatment groups, female subjects displayed faster pharmacokinetics in comparison to male subjects, although not reaching significance. We conclude that UPLC-MS/MS is the method of choice to measure trough concentrations of DOACs in patients after orthopaedic surgery. Current coagulation assays are not suited for this purpose. We found large heterogeneity in both peak and trough concentrations of DOACs, and showed that pharmacokinetics of novel oral anticoagulants may be influenced by age and gender. Whether patients with high or low trough concentrations are at increased risk for bleeding or thromboembolic events respectively remains to be established.

Hyponatraemia in elderly emergency department patients: a marker of frailty.

BACKGROUND: Details on hyponatraemia in the emergency department are limited, especially regarding older patients, a population more susceptible to hyponatraemia and its effects. Our objective was to gain insight into the prevalence, aetiology, treatment and prognosis of clinically relevant hyponatraemia in elderly emergency department patients. The impact of the severity of hyponatraemia on outcome was a secondary objective. METHODS: A retrospective cohort study of 1438 internal medicine patients aged ≥ 65 years presenting to the emergency department between 1 September 2010 and 31 August 2011 was performed. Clinically relevant hyponatraemia was defined as a serum sodium level < 130 mmol÷l. The reference group had a serum sodium level of 130-145 mmol÷l. Hyponatraemia was subdivided into moderate (129-125 mmol÷l), and severe (< 125 mmol÷l). RESULTS: Ninety-one elderly patients (6.3%) were hyponatraemic at presentation to the emergency department. The main causes were the use of diuretics, hypovolaemia, and the syndrome of inappropriate antidiuretic hormone secretion (57.1%). Hyponatraemia was associated with higher admission rates (93.4 vs. 72.9%) and longer hospital stay (8 vs. 6 days) vs. the reference group. Three-month survival rate in hyponatraemic elderly patients was 74% (95% CI 64-84%) vs. 83% (95% CI 81-85%) in the reference group. Moderate hyponatraemia was associated with an increased risk of death (HR 1.7, 95% CI 1.2-2.4) vs. the reference group after multivariable adjustment for age and comorbidity. CONCLUSION: Hyponatraemia, a common electrolyte disturbance among elderly internal medicine patients presenting to the emergency department, was associated with higher admission rates, longer hospital stay, and higher mortality rates. In particular, moderate hyponatraemia was a marker of underlying frailty and predictive of mortality.

Tolbutamide stimulation of pancreatic beta-cells involves both cell recruitment and increase in the individual Ca(2+) response.

Individual pancreatic beta-cells are functionally heterogeneous. Their sensitivity to glucose is variable, so that the proportion of active cells increases with the glucose concentration (recruitment). We have investigated whether sulphonylureas also recruit beta-cells, by measuring cytoplasmic Ca(2+) ([Ca(2+)](i)) - the triggering signal of insulin secretion - in single cells and clusters of cells prepared from mouse islets. In 4 mM glucose, the threshold concentration of tolbutamide inducing a [Ca(2+)](i) rise was variable (5 - 50 microM). The proportion of responsive cells and clusters therefore increased with the tolbutamide concentration, to reach a maximum of 90% of the cells and 100% of the clusters. This recruitment occurred faster when the glucose concentration was increased from 4 to 5 mM (EC(50) of approximately 14 and approximately 4 microM tolbutamide respectively). Within responsive clusters little recruitment was observed; when a cluster was active, all or nearly all cells were active probably because of cell coupling. Thus, tolbutamide-induced [Ca(2+)](i) oscillations were synchronous in all cells of each cluster, whereas there was no synchrony between clusters or individual cells. Independently of cell recruitment, tolbutamide gradually augmented the magnitude of the [Ca(2+)](i) rise in single cells and clusters. This increase occurred over a broader range of concentrations than did recruitment (EC(50) of approximately 50 and 25 microM tolbutamide at 4 and 5 mM glucose respectively). Tolbutamide (10 microM) accelerated the recruitment of single cells and clusters brought about by increasing glucose concentrations (range of 3 - 7 mM instead of 4 - 10 mM glucose), and potentiated the amplification of the individual responses that glucose also produced. In conclusion, both metabolic (glucose) and pharmacologic (sulphonylurea) inhibition of K(+)-ATP channels recruits beta-cells to generate a [Ca(2+)](i) response. However, the response is not of an all-or-none type; it increases in amplitude with the concentration of either glucose or tolbutamide.

Measurements of cytoplasmic Ca2+ in islet cell clusters show that glucose rapidly recruits beta-cells and gradually increases the individual cell response.

The proportion of isolated single beta-cells developing a metabolic, biosynthetic, or secretory response increases with glucose concentration (recruitment). It is unclear whether recruitment persists in situ when beta-cells are coupled. We therefore measured the cytoplasmic free Ca2+ correction ([Ca2+]i) (the triggering signal of glucose-induced insulin secretion) in mouse islet single cells or clusters cultured for 1-2 days. In single cells, the threshold glucose concentration ranged between 6 and 10 mmol/l, at which concentration a maximum of approximately 65% responsive cells was reached. Only 13% of the cells did not respond to glucose plus tolbutamide. The proportion of clusters showing a [Ca2+]i rise increased from approximately 20 to 95% between 6 and 10 mmol/l glucose, indicating that the threshold sensitivity to glucose differs between clusters. Within responsive clusters, 75% of the cells were active at 6 mmol/l glucose and 95-100% at 8-10 mmol/l glucose, indicating that individual cell recruitment is not prominent within clusters; in clusters responding to glucose, all or almost all cells participated in the response. Independently of cell recruitment, glucose gradually augmented the magnitude of the average [Ca2+]i rise in individual cells, whether isolated or associated in clusters. When insulin secretion was measured simultaneously with [Ca2+]i, a good temporal and quantitative correlation was found between both events. However, beta-cell recruitment was maximal at 10 mmol/l glucose, whereas insulin secretion increased up to 15-20 mmol/l glucose. In conclusion, beta-cell recruitment by glucose can occur at the stage of the [Ca2+]i response. However, this type of recruitment is restricted to a narrow range of glucose concentrations, particularly when beta-cell association decreases the heterogeneity of the responses. Glucose-induced insulin secretion by islets, therefore, cannot entirely be ascribed to recruitment of beta-cells to generate a [Ca2+]i response. Modulation of the amplitude of the [Ca2+]i response and of the action of Ca2+ on exocytosis (amplifying actions of glucose) may be more important.

Prevalence and content analysis of guidelines on handling requests for euthanasia or assisted suicide in Dutch nursing homes.

BACKGROUND: The growing number of requests for euthanasia or assisted suicide (EAS) makes it imperative for health care institutions, such as nursing homes, to have written guidelines on how to handle requests for EAS. The objective of this study was to determine the prevalence of EAS guidelines in Dutch nursing homes and to analyze the content. METHODS: Directors of patient care in 324 Dutch nursing homes were asked, by means of a mailed short list of questions, if they had an institutional guideline on EAS and, if so, to provide a copy. Guidelines were analyzed according to a structured list of items based on current jurisprudence, model documents, and opinions of experts. RESULTS: Of the 324 directors, 313 (97%) responded. In 58% of the nursing homes that responded, there existed written guidelines for EAS. Of those guidelines, 74% concerned EAS; in 26%, EAS was integrated in a guideline on terminal care. Of the guidelines, 165 (90%) were based on the policy that EAS is acceptable under specific conditions, and 18 (10%) banned EAS completely. Of the first-mentioned guidelines, 81% described one or more procedures for in-principle objections. In 65% of these guidelines, all official requirements for prudent practice were described. CONCLUSIONS: Despite the rapidly growing number of nursing-home guidelines on EAS and the existence of model documents, there is still considerable variation in the guidelines, and they can be improved in many aspects. A basic prerequisite is that the guidelines include all the official requirements for prudent practice.

The oscillatory behavior of pancreatic islets from mice with mitochondrial glycerol-3-phosphate dehydrogenase knockout.

Glucose stimulation of pancreatic beta cells induces oscillations of the membrane potential, cytosolic Ca(2+) ([Ca(2+)](i)), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca(2+) have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca(2+)](i). In the present study, we compared different types of oscillations in beta cells from wild-type and mGPDH(-/-) mice. In clusters of 5-30 islet cells and in intact islets, 15 mM glucose induced an initial drop of [Ca(2+)](i), followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca(2+)] rise, were similar in wild-type and mGPDH(-/-) mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH(-/-) beta cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca(2+)](i) during imposed oscillations induced by pulses of high K(+) or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca(2+)](i) in beta cells similarly increased NAD(P)H fluorescence in control and mGDPH(-/-) islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca(2+)](i) and secretory responses in mGPDH(-/-) islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of beta cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca(2+).

Influence of cell number on the characteristics and synchrony of Ca2+ oscillations in clusters of mouse pancreatic islet cells.

1. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single cells and cell clusters of different sizes prepared from mouse pancreatic islets. 2. During stimulation with 15 mM glucose, 20 % of isolated cells were inert, whereas 80 % showed [Ca2+]i oscillations of variable amplitude, duration and frequency. Spectral analysis identified a major frequency of 0.14 min-1 and a less prominent one of 0.27 min-1. 3. In contrast, practically all clusters (2-50 cells) responded to glucose, and no inert cells were identified within the clusters. As compared to single cells, mean [Ca2+]i was more elevated, [Ca2+]i oscillations were more regular and their major frequency was slightly higher (but reached a plateau at approximately 0.25 min-1). In some cells and clusters, faster oscillations occurred on top of the slow ones, between them or randomly. 4. Image analysis revealed that the regular [Ca2+]i oscillations were well synchronized between all cells of the clusters. Even when the Ca2+ response was irregular, slow and fast [Ca2+]i oscillations induced by glucose were also synchronous in all cells. 5. In contrast, [Ca2+]i oscillations resulting from mobilization of intracellular Ca2+ by acetylcholine were restricted to certain cells only and were not synchronized. 6. Heptanol and 18alpha-glycyrrhetinic acid, two agents widely used to block gap junctions, altered glucose-induced Ca2+ oscillations, but control experiments showed that they also exerted effects other than a selective uncoupling of the cells. 7. The results support theoretical models predicting an increased regularity of glucose-dependent oscillatory events in clusters as compared to isolated islet cells, but contradict the proposal that the frequency of the oscillations increases with the number of coupled cells. Islet cell clusters function better as electrical than biochemical syncytia. This may explain the co-ordination of [Ca2+]i oscillations driven by depolarization-dependent Ca2+ influx during glucose stimulation.

Collaborative study on the use of motility enrichment on modified semisolid Rappaport-Vassiliadis medium for the detection of Salmonella from foods.

A collaborative study was performed in 15 laboratories to evaluate the use of motility enrichment on modified semisolid Rappaport-Vassiliadis (MSRV) medium for rapid Salmonella detection in a variety of food products. The results of this procedure were compared with those obtained by the cultural procedure using Rappaport-Vassiliadis (RV) broth as selective enrichment and modified brilliant green agar for selective plating. The tests were performed with Salmonella reference samples (SRS) as well as with naturally contaminated food products. When SRS were used without added food the productivity of both MSRV and RV was 96%. When SRS were combined with reference samples containing competitive bacteria the productivity was 98% for MSRV and 95% for RV. In the tests with food samples the productivity of MSRV was 92% with SRS added to food and 96% with naturally contaminated samples, while the productivity of RV was 88% and 90%, respectively. Statistical analysis showed that there was no significant difference between the procedures.

A comparative study of suriclone, lorazepam and placebo in anxiety disorder.

In a four-week double-blind randomized trial preceded by a one-week single-blind placebo treatment, the efficacy and the side-effects of suriclone (1.50-2.25 mg/d) (n = 24), lorazepam (5.0-7.5 mg/d) (n = 19) and placebo (n = 21) were compared in 64 outpatients with a DSM-III diagnosis of generalized anxiety disorder (n = 56) or panic disorder (n = 8). Efficacy was measured weekly by means of a global clinical impression scale, the Hamilton Anxiety Rating Scale, the Zung Anxiety Self-Assessment Scale and a target symptom scale. Side-effects were evaluated weekly by an adverse events scale, which recorded the spontaneous complaints and the complaints elicited by an extensive somatic inventory questionnaire. The three groups showed a statistically significant and clinically relevant improvement early on in the treatment: this improvement was maintained during the remaining period. Early on in the treatment there was some indication of a better response, but also of more side-effects, in the suriclone and the lorazepam groups. After four weeks of treatment no difference was found between the three groups either in efficacy or in side-effects. The effect size achieved in the placebo group was not inferior to that of benzodiazepines in general.

Flurazepam and temazepam in the treatment of insomnia in a general hospital population.

This study is a double-blind comparative trial of flurazepam and temazepam in the treatment of insomnia, using subjective assessments with an analogue scale technique and questionnaire. The main dependent variable in this experiment is vigilance. The two drugs differ essentially in their half life value. (Flurazepam +/- 72 h; temazepam +/- 8 h). It can be predicted that temazepam causes less impairment of vigilance than flurazepam, and that the difference between the two drugs is more pronounced when the number of days the drugs are taken consecutively, increases. In statistical terms an interaction effect between drugs and days should be expected. The results show no effect at all, neither the predicted interaction effect, nor any main effect. If there is a difference in efficiency reduction between patients, the assessment methods used were unable to measure it. These findings warn against a possible overestimation of clinical relevance of the plasma elimination half-life of benzodiazepines.